E.—Action of Various Diastases upon Venoms.
Venoms are modified or destroyed by certain normal diastases of the organism. It was shown long ago by Lacerda, Weir Mitchell, Sir Joseph Fayrer, and Lauder Brunton, that it is possible to introduce without danger into the stomachs of adult animals amounts of venom many times greater than the lethal dose. I have repeatedly verified this, but have nevertheless observed that young mammals, while being suckled, readily absorb venom by their alimentary canal, and succumb to the ingestion of doses scarcely larger than those which kill when subcutaneously injected. Here we have a very important fact, which once more proves how easily the intestinal mucous membrane of young animals is permeated by toxins. By my instructions Wehrmann[64] and Carrière,[65] in my laboratory, have studied the modifications undergone by venoms in the alimentary canal of rabbits. We have seen that these animals can ingest without inconvenience doses of venom 600 times greater than the lethal dose, and that, if we cause these ingestions to be repeated several times, contrary to the assertion of Fraser[66] (of Edinburgh), we never succeed in obtaining immunity to the subcutaneous injection of a simple lethal dose, and no antitoxin is formed in the blood.
The ptyalin of the saliva, pancreatic juice, and bile destroy Cobra-venom in vitro. We must, therefore, assume that these diastases are veritable agents of destruction for ingested venom. The intestinal microbes play no part, any more than the intestinal juice by itself. The gastric juice has very little effect. Papain is almost as active as the pancreatic juice.
It had already been proved by Fraser, so long ago as 1895, that bile, after prolonged contact and in a sufficient dose, has a strongly destructive effect upon Cobra-venom; but, contrary to the opinion of this investigator, it is not antitoxic, for it possesses no preventive or curative property, and its effects are produced only in vitro.
We see from what has been stated above, that venoms introduced into a sensitive organism are capable of producing extremely complex effects upon the various tissues or humours. They act on the nerve-cells by their neurotoxin, on the endothelium of the blood-vessels by their hæmorrhagin (Flexner and Noguchi), on the red corpuscles by their hæmolysin, on the fibrin of the blood and muscles by their proteolytic diastase, and on the fibrin-ferment itself by their thrombase.
They also act on the leucocytes, according to the experiments of Chatenay,[67] performed under the direction of Metschnikoff, and according to those of Flexner and Noguchi,[68] already cited.
Thus we understand how complex must likewise be the means of defence that have to be employed in order to afford an effective protection against such poisons.
The slightly intoxicated organism at first reacts by the intervention of the leucocytes; a hyperleucocytosis is produced, accompanied by a more or less considerable rise of temperature. After a few hours everything returns to its normal condition, and if the injection of a lethal dose of venom is repeated several times, at intervals of a few days, it is not long before antitoxic substances are found to appear in the serum.
When the dose of venom injected is sufficient to cause death, we observe, a very few moments after the injection, a lowering of temperature and a hypoleucocytosis, which is the more pronounced in proportion to the nearness of the dose of venom to the minimal lethal dose. With very strong doses the hypoleucocytosis has not time to manifest itself.
It is therefore probable that, in intoxication by venoms as in that by the toxins of micro-organisms, the protective rôle of the leucocytes is all-important, not only because these cells are capable of digesting venoms owing to their protoplasmic digestive juices, but also because they constitute if not the only, at any rate the principal source of the antitoxic substances or amboceptors.
CHAPTER X.
TOXICITY OF THE BLOOD OF VENOMOUS SNAKES.
Several physiologists, among whom it is right to mention Fontana,[69] Leydig,[70] Reichel,[71] Raphael Blanchard,[72] Phisalix and Bertrand,[73] and S. Jourdain,[74] have pointed out the presence of poison-glands in Tropidonotus natrix or other non-venomous snakes, and have explained the immunity enjoyed by these animals with regard to venom as being due to the existence of an internal secretion of this poison.
We also know, from the writings of Phisalix and Bertrand, that the blood of the viper, and that of the salamander and toad are toxic. For my part I have found[75] that the blood of Naja, Bungarus, Lachesis, and Cerastes possesses the same properties, and a comparative study has been made by Wehrmann,[76] in my laboratory, of the toxicity of the blood of the viper and of that of the blood of the eel, already established by Mosso (of Turin).[77]
It is remarkable to find that the blood of the various venomous or non-venomous snakes, like that of certain fishes, such as eels, produces, when injected beneath the skin or into the peritoneum, local and general effects very similar to those of venoms. Injections of 0·5 c.c. to 1 c.c. of the blood of the viper or of the common snake, beneath the skin of the guinea-pig, provoke an intense local reaction, which always results in the formation of an eschar. The injection of slightly stronger doses, 1 c.c. to 2 c.c., into the peritoneum, almost always kills these animals, like venom, with symptoms of respiratory asphyxia.
The blood of Naja tripudians, injected subcutaneously, is lethal to the mouse in a dose of 0·25 c.c.
When this blood is heated, after having been suitably diluted with three or four parts of distilled water, in order to prevent it from coagulating, it is found that a temperature of 70° C. maintained for fifteen minutes is sufficient to cause it to lose all toxic effect. The same applies to the blood of the other poisonous or non-poisonous snakes, and to that of the Murænidæ.
Now, since the majority of venoms resist even prolonged heating at this temperature, it cannot be supposed that the toxicity of the blood is due to its containing venom derived from the internal secretion of the poison-glands, as was thought by Phisalix and Bertrand. On the contrary, it is probable that the toxicity results from the fact that the blood contains diastasic substances of cellular origin, which themselves represent certain of the constituent elements of venoms.
These substances, moreover, possess some of the properties of venoms, as, for instance, the faculty of producing hæmorrhages and of being influenced by antivenomous serum, which causes them to lose a large portion of their toxic qualities.
I have found that they can even be utilised to vaccinate animals against venom; by injecting weak, non-lethal, and repeated doses of dilute Cobra-blood into guinea-pigs and rabbits, I have succeeded in rendering them immune to doses of Cobra-venom several times greater than the lethal dose.
There is no doubt that it is to these substances that the poisonous and non-poisonous snakes owe the partial immunity that they themselves enjoy with respect to venoms. We know, in fact, that common snakes suffer without danger many bites from vipers (Phisalix and Bertrand[78]), and that the Cobra is relatively little affected by inoculation with its own venom or with that of other Colubridæ, such as Bungarus, or even of Viperidæ, such as Vipera russellii.
This immunity, however, is far from being absolute; I have killed common snakes (Tropidonotus natrix) with doses of viper-venom ten times greater than the lethal one for the rabbit, and Lachesis lanceolatus (from Martinique) with 0·02 gramme of the venom of Naja tripudians.
Phisalix,[79] on his part, has shown that, while it was necessary to inject from 100 to 200 milligrammes of viper-venom into other vipers or common snakes, beneath the skin or into the peritoneum, in order to cause death, the introduction of only 2 to 4 milligrammes of this venom into the brains of these reptiles was sufficient to kill them with the same symptoms of intoxication. This dose, however, is only twenty-five to thirty times greater than the lethal one for the guinea-pig.
The practical lesson to be learnt from the establishment of the foregoing facts is that poisonous snakes of different species must never be placed in the same cage, for these animals sometimes bite each other, and may thus kill one another.
Simon Flexner and Noguchi[80] have studied the action of the serums of Crotalus, Ancistrodon, and a non-poisonous species, the pine snake (Pituophis catenifer), on the venoms of Naja, Ancistrodon, and Crotalus. They found that the serum of Crotalus rapidly dissolves the red corpuscles of man, the dog, rabbit, guinea-pig, sheep, rat, pigeon, and horse.
The serum of the pine snake affects the same red corpuscles, but in a lesser degree. Heating to 58° C. suppresses the hæmolytic power of these serums, but they can be restored to activity by the addition of a very small quantity of the same serum in a fresh condition, of fresh serum derived from other snakes, or of fresh serum from the guinea-pig.
Antivenomous serum also, when added in a suitable dose, entirely suppresses the hæmolytic action of snake-serums; it has, however, greater effect upon the hæmolysin of Cobra-blood than upon that of the blood of other snakes. This observation had previously been made by W. Stephens,[81] and it has been verified by Noc in my laboratory.
Crotalus-serum dissolves the red corpuscles of the mongoose (Herpestes ichneumon) of Jamaica, whose extraordinary resistance to venom is well known. But if variable doses of Ancistrodon-venom and Crotalus-serum be made to act simultaneously upon these corpuscles, the latter are no longer dissolved. Again, if, instead of red corpuscles which are but little sensitive, like those of the mongoose, we employ the highly sensitive corpuscles of the guinea-pig, the result is the same. These experiments are regarded by Flexner and Noguchi as proving that the amboceptors of the toxic serum become fixed, in conformity with Ehrlich’s theory of the lateral chains, upon the receptors of the sensitive erythrocytes, and leave no more receptors free for the fixation of the venom.
The same investigators have endeavoured to determine the respective toxicity of the tissues of the different organs of Crotalus. They found that the most toxic organs are the spleen and the liver; the toxicity of the spinal cord, kidney and muscles is much less. It appears that this toxicity is intimately connected with the quantity of blood that the tissues retain, for the physiological effects observed are identical with those that follow the injection of blood or serum alone.
They also ascertained that the contents of the eggs of Crotalus are especially rich in poison, and this poison appears to consist for the most part of neurotoxin, since it does not cause hæmorrhages. Phisalix has observed that the ovules of the viper exhibit analogous toxicity.[82]
Summing up what has been stated above, we find that the blood of both poisonous and non-poisonous snakes contains toxic substances, destructible by heating to 68° C., and physiologically distinct from venoms, but like the latter possessing the property of dissolving the red corpuscles of the majority of vertebrates and of producing hæmorrhages.
CHAPTER XI.
NATURAL IMMUNITY OF CERTAIN ANIMALS WITH RESPECT TO SNAKE-VENOMS.
It was long ago pointed out that certain warm-blooded animals, including the mongoose (Herpestes ichneumon, Family Viverridæ), hedgehog (Erinaceus europæus, Family Erinaceidæ), pig (Sus scrofa, Family Suidæ), and some herons (Ajaja, Subfamily Plataleinæ; Cancroma, Subfamily Cancrominæ; Botaurus, Subfamily Ardeinæ; Mycteria, Subfamily Ciconiinæ), known in Colombia under the names Culebrero and Guacabo, exhibit a natural immunity with respect to snake-bites.
Pigs devour vipers with great readiness, and in the region of North America which adjoins the Mississippi and its tributaries they are even trained to destroy the young rattle-snakes and other poisonous serpents with which the valleys of these watercourses are infested.
During my stay in Indo-China I inoculated a young pig, beneath the skin of the back, with a dose of Cobra-venom (10 milligrammes) capable of killing a large-sized dog. The animal withstood the injection, but I am inclined to think that this is not a case of true immunity; it is probable that the pig owes its resistance to venom to the fact that its skin is lined with an enormous layer of adipose tissue, which is but very slightly vascular, and in which absorption takes place very slowly. This opinion is corroborated by my discovery that the serum of this animal is entirely destitute of any antitoxic substance. I mixed a dose of Cobra-venom, lethal for the rabbit, with 3·5 and 8 c.c. of pig-serum. These mixtures killed rabbits in the same time as the controls that received the venom diluted with equal quantities of rabbit-serum or physiological saline solution.
The natural immunity of the mongoose and the hedgehog rests upon more scientifically established facts.
My own experiments upon the immunity of the mongoose were made with six specimens of these little carnivores captured in Guadeloupe (French West Indies), an island in which no poisonous snakes exist; consequently their immunity could not have arisen from their having become accustomed to the bites of venomous reptiles.
I first introduced a mongoose into a cage containing a Naja bungarus (Ophiophagus) of large size. The snake rose up immediately, dilated its hood, and struck savagely at the little animal, which, darting nimbly out of the way, escaped being seized and, frightened for a moment, took refuge in a corner of the cage. Its stupor, however, was but of brief duration, for at the very moment when the hamadryad was preparing to strike at it again, the mongoose, with open mouth and snarling, sprang upon the reptile’s head, bit it hard in the upper jaw and crushed its skull in a few seconds. This scene is in every respect reminiscent of the admirable description given by Rudyard Kipling, in his celebrated “Jungle Book,” of the great war that Rikki-tikki (the Mongoose) fought with Nag (the Cobra) “through the bathrooms of the big bungalow in Segowlee cantonment”:—
“Nag was asleep, and Rikki-tikki looked at his big back, wondering which would be the best place for a good hold. ‘If I don’t break his back at the first jump,’ said Rikki, ‘he can still fight; and if he fights—O Rikki!’ He looked at the thickness of the neck below the hood, but that was too much for him; and a bite near the tail would only make Nag savage.
“’It must be the head,’ he said at last; ‘the head above the hood; and when I am once there, I must not let go.’
“Then he jumped. The head was lying a little clear of the water-jar, under the curve of it; and, as his teeth met, Rikki braced his back against the bulge of the red earthenware to hold down the head. This gave him just one second’s purchase, and he made the most of it. Then he was battered to and fro as a rat is shaken by a dog—to and fro on the floor, up and down, and round in great circles; but his eyes were red, and he held on as the body cart-whipped over the floor, upsetting the tin dipper and the soap-dish and the flesh-brush, and banged against the tin side of the bath. As he held he closed his jaws tighter and tighter, for he made sure he would be banged to death, and, for the honour of his family, he preferred to be found with his teeth locked. He was dizzy, aching, and felt shaken to pieces, when something went off like a thunderclap just behind him; a hot wind knocked him senseless, and red fire singed his fur. The big man had been wakened by the noise, and had fired both barrels of a shot-gun into Nag just behind the hood.”[83]
From the experimental point of view, these stirring battles between mongooses and cobras only show that a mongoose of the size of a large squirrel makes a plucky and victorious attack upon a venomous reptile of the most dangerous species and of very large dimensions; but it is impossible to tell with certainty whether the mongoose has been bitten.
I therefore inoculated a second mongoose with 2 milligrammes of venom, a lethal dose for 4 kilogrammes of rabbit. The animal did not experience the slightest malaise.
I then took blood from three other mongooses, by tying a carotid without killing the animals. This blood, mixed with venom or injected as a prophylactic into rabbits, exhibited an antitoxic power, which, though evident, was of little intensity, and insufficient in all cases as a certain preventative of death. All the rabbits that received a preventive dose varying from 2 to 7 c.c. of mongoose-serum succumbed to inoculation with venom, but with a considerable retardation (from two to five hours) as compared with the controls.
I endeavoured to determine the limit of tolerance of the mongoose with respect to venom. Two of these animals, which had never been inoculated, received doses of venom respectively four times and six times lethal for the rabbit. The first mongoose remained perfectly well; the second was ill for two days, and then recovered. A third mongoose, into which I injected a dose eight times lethal for the rabbit, succumbed in twelve hours.
Fig. 89.—Mongoose seized by a Cobra.
(For this illustration I am indebted to the kindness of M. Claine, late French Consul at Rangoon.)
It must be concluded from these facts that the West Indian mongoose is but little sensitive to venom; that it is capable of withstanding, without malaise, doses which are considerable in proportion to its size, but that its immunity is far from being absolute. If it is generally the victor in its combats with poisonous snakes, the result is mainly due to the extreme agility with which it is endowed.
A number of experiments have been made by Lewin,[84] and by Phisalix and Bertrand,[85] upon the immunity of the hedgehog to the venom of Vipera berus.
It had long been known that hedgehogs are inveterate hunters of vipers, which they devour with avidity. Thanks to the long and sharp spines by which their bodies are protected, they avoid being bitten and contrive to catch the reptiles very cleverly, but it occasionally happens that they do not escape being struck. However, even in these cases they rarely succumb.
Inoculation with fairly large quantities of venom does not make them ill: the dose of viper-venom lethal for these small animals is about forty times greater than that which kills the guinea-pig. Their power of resistance is therefore beyond doubt.
It may be asked whether this is due to their blood normally containing antitoxic substances. In order to elucidate this question, Phisalix and Bertrand first proved that the blood of normal hedgehogs is toxic to laboratory animals, especially to the guinea-pig. A mixture of this blood with viper-venom cannot therefore be harmless. But it sufficed to heat hedgehog blood to 58° C. to cause it to lose its toxicity, and it was found that it then became antitoxic. Guinea-pigs inoculated in the peritoneum with 8 c.c. of heated hedgehog-serum were able to withstand, immediately afterwards, twice the lethal dose of viper-venom.
It really seems, therefore, that the resistance of the hedgehog to venom is due to the presence of antitoxic substances in its blood. But, as in the case of the mongoose, there is no question here of genuine immunity.
The same is probably true with respect to the herons of Colombia, the Culebrero and Guacabo, which eagerly search after young snakes for food. No investigations, however, have yet been made upon this subject.
These birds, moreover, are few in number; hunters pursue them for the sake of their brilliantly coloured plumage, and it is to be regretted that no attempt is made to prevent their destruction or to acclimatise them in countries in which poisonous snakes constitute a veritable calamity, such as Martinique, St. Lucia, or India.
CHAPTER XII.
SNAKE-CHARMERS.
In all the countries of the globe where poisonous snakes are formidable to man, there are certain individuals who profess to be secure from all ill-effects from the bites of these reptiles, whether because they are immune to venom, or because they possess secrets which enable them to cure themselves when they happen to have been bitten. Not unnaturally these secrets are sometimes turned to profitable account, and the possessors of them generally enjoy considerable popular influence, and are very highly venerated. Intimate relations with the divinities are freely attributed to them.
Among the Romans the jugglers who carried on the profession of snake-charmers and healers of snake-bites were known as Psylli. Plutarch tells us that Cato, who loved not doctors because they were Greeks, attached a certain number of them to the army of Libya. They were accustomed to expose their children to serpents as soon as they were born, and the mothers, if they had failed in conjugal fidelity, were infallibly punished by the death of their offspring. If, on the contrary, the children were lawful, they had nothing to fear from the bites of the reptiles. “Recens etiam editos serpentibus offerebant; si essent partus adulteri, matrum crimina plectabantur interitu parvulorum; si pudici, probos ortus a morte paterni privilegium tuebatur” (Solinus).
The Libyian Psylli of antiquity still have their representatives in Tunis and in Egypt. Clot Bey writes as follows with reference to the Egyptian Psylli:—
“The Ophiogeni, or Snake-charmers, have been renowned from all time. Strabo speaks of them, and Prosper Alpinus was a witness of the singular effects of their art. The majority of modern travellers who have visited Egypt have been equally struck with the freedom with which they handle poisonous reptiles and animals.
“The Psylli go from house to house, calling forth and charming the snakes that they may happen to contain. They claim to attract them by means of a particular power. Armed with a short wand, they enter the chamber to be purged from these venomous guests, make a smacking noise with their tongue, spit upon the ground, and pronounce the following incantation: ‘I adjure you, by God, if you are without or within, to appear; I adjure you, by the greatest of names; if you are obedient, appear! If you disobey, die! die!’ The snake, submissive to this command, departs forthwith, issuing from a crack in the wall or floor.”[86]
India is pre-eminently the country of snake-charmers. There exists an entire caste of Hindus, called Mal, who are professional catchers and vendors of snakes, but do not perform tricks with them.
The snake-charmers are recruited from among another caste, that of the Sangis or Tubriwallahs of Bengal.
These men, who are usually clothed in yellow robes and wear large turbans, manage the Cobra with really marvellous skill. All travellers who have had the opportunity of crossing India or of touching at a port on the coast or on that of Ceylon have witnessed scenes similar to that described by Natalis Rondot (figs. 90 and 91):—
“Towards six o’clock in the evening a Hindu juggler comes on board. He is poorly clad, and wears a turban decorated with three feathers, and several necklaces of those amulet-sachets called gris-gris in Senegal. In a flat basket he carries a spectacled Cobra-di-Capello.
Fig. 90.—Indian Snake-charmer at Colombo (Ceylon).
Fig. 91.—Indian Snake-charmer at Colombo (Ceylon).
“This man instals himself on deck; we sit down on the seat provided for the officer of the watch, and the sailors form a circle. The basket is placed on the deck and uncovered; the Cobra is coiled up at the bottom of it. The juggler squats a few paces off and commences to play a slow, plaintive, and monotonous air, with a kind of small clarinet ([fig. 92]), the sounds of which recall those of the Breton biniou.
Fig. 92.—Musical Instrument used by Indian Snake-charmers to Charm Cobras.
(For this figure I am indebted to the kindness of Dr. Pineau.)
“By degrees the snake moves, extends itself, and then assumes an erect posture, but without quitting the basket. It begins to appear uneasy and endeavours to recognise its surroundings; it becomes agitated and irritated, expands and spreads out its hood, breathes hard rather than hisses, and frequently and quickly shoots out its slender forked tongue; several times it makes a violent dart as though to attack the juggler; it frequently trembles, or rather gives sudden starts. The juggler keeps his eyes always fastened upon the snake, and gazes at it with a singular fixed stare. After some time, about ten or twelve minutes, the Cobra becomes less animated, grows quiet, and then sways as though influenced by the slow and monotonous rhythm of the musician; it keeps incessantly darting out its tongue. Little by little it is brought to a sort of somnolent condition. Its eyes, which at first watched the juggler as though in order to take him by surprise, are, to a certain extent, fixed and fascinated by the latter’s gaze. The Hindu takes advantage of this moment of stupefaction on the part of the snake by approaching it slowly without ceasing to play, and touches the head of the Cobra, first with his nose and then with his tongue. Although this takes but an instant the reptile starts out of its sleep, and the juggler has barely time to throw himself backwards so as not to be struck by the snake, which makes a furious dart at him.
“We doubt whether the Cobra still has it fangs, and whether the Hindu incurs any real danger in approaching it. Accordingly we promise our man a Spanish piastre if he will make the snake bite a couple of fowls. A black hen, which struggles violently, is taken and offered to the Cobra, which half rises, looks at the bird, bites it, and lets it go. The fowl is released and runs off terrified. Six-minutes later, by the watch, it vomits, stretches out its legs, and dies. A second fowl is placed in front of the snake, which bites it twice, and the bird dies in eight minutes.”[87]
Certain jugglers exhibit snakes from which they have taken care to extract the fangs; they offer the animal a piece of cloth or soft stuff into which it drives its poison-teeth, and the fabric is then quickly snatched away in order by this means to break off the poison-fangs that have penetrated it. This operation is repeated at certain intervals with a view to preventing the reserve fangs from coming into use, and the reptiles can then be handled without any danger.
It is unquestionable, however, and I have personally satisfied myself of the fact, that many genuine snake-charmers go through their performances with Cobras whose poison-apparatus is absolutely intact. That they almost always avoid being bitten is due to a perfect knowledge of the habits and movements of these reptiles. Nevertheless, accidents sometimes happen to them, and every year a few of them succumb in pursuit of their calling (see p. 370). Still, it may be asserted that some of them really know how to vaccinate themselves against venom, by making young Cobras bite them from time to time.
It is stated by E. C. Cotes,[88] formerly of the Calcutta Museum, that the Indian snake-charmers do not extract the poison-fangs from their snakes. Even though deprived of its fangs, the snake would still be dangerous on account of its other teeth, the punctures of which would provide another channel for the penetration of the venom.
Snake-charmers pretend that they owe their immunity to graduated inoculations. This is not yet conclusively proved; what is better established is that they take the greatest care to avoid being bitten, and that in so doing they display the most remarkable skill.
Even in France we are acquainted with professional viper-catchers, who employ the method of graduated inoculations in order to render themselves immune to the bites of indigenous reptiles. One of these men, who lives near Arbois (Jura), takes good care to get himself bitten, at least once a year, by a young viper; when he forgets this precaution and happens to be bitten, he always feels the effects much more severely.
Fraser[89] (of Edinburgh) thinks that the repeated ingestion of small quantities of venom may suffice to confer immunity, and he mentions a certain number of experiments performed by him upon white rats and kittens, from which it would appear that the ingestion of venom, continued for a long time, finally renders these animals absolutely refractory to subcutaneous inoculation with doses of the same venom several times greater than the lethal one. He therefore concludes that this process of vaccination may probably be in use among snake-charmers.
I have submitted this hypothesis to the test of experiment. I succeeded in making adult rabbits, guinea-pigs, and pigeons absorb enormous doses of Cobra-venom by way of the alimentary canal. In this manner I have administered doses as much as a thousand times greater than the lethal one, yet I have never been able to prove that the serum of these animals became antitoxic.
On the other hand, I have succeeded in vaccinating very young guinea-pigs and young rabbits which were still being suckled, by making them absorb, every second day, minimal and certainly innocuous doses of very dilute venom. In the case of young animals, venom is not modified by the digestive juices, and a portion of it is absorbed by the mucous membrane of the intestine. When the dose ingested is suitably reduced they withstand it, and when these ingestions are repeated every second or third day during the first weeks of life, the animals become perfectly vaccinated against doses certainly lethal for controls of the same age and weight. But it is always difficult to push the vaccination far enough for the serum to acquire antitoxic properties, and I have never been able to prove the appearance of the latter.
I think, however, that it ought to be possible to arrive at this result by experimenting upon animals such as lambs, kids, calves, or foals, the intestine of which remains permeable to toxins for a sufficiently long period.
It may be that certain snake-charmers, who claim to possess family secrets which they transmit from father to son, employ an analogous method in order, in their infancy, to confer immunity to venoms upon those of their male children who are to inherit their strange and lucrative profession.
In Mexico, certain Indians called Curados de Culebras know how to acquire the privilege of being able to be bitten by poisonous snakes without the least danger to life, by inoculating themselves several times with the teeth of rattle-snakes.
Dr. Jacolot,[90] a naval surgeon, while staying at Tuxpan, made enquiries as to these Curados de Culebras, and was able to satisfy himself that their immunity is an actual fact.
The process of vaccination employed by the natives of Tuxpan is as follows:—A preparatory treatment is necessary. On the very day on which a man is to inoculate himself or get himself inoculated, he takes from 5 to 15 tubers of a plant known by the name of Mano de Sapo (i.e., Toad’s hand, Dorstenia contrayerva, Family Urticaceæ). These tubers must—and this is absolutely necessary—be administered on a Friday, and always in an odd number, 5, 7, 9, &c., up to 15, according to the tolerance of the subject.
If the plant be gathered on the first Friday in March it possesses its marvellous properties in the highest degree; in this case, even if it be dry, it is still excellent for the preparatory stage of the inoculation.
The physiological effects of mano de sapo are not very marked: the circulation is slightly diminished and a sensation of cold is experienced, but there are no nervous troubles. The subject frequently has attacks of vomiting or nausea. The inclination to vomit must be fought against, for if the plant should happen to be rejected it would be dangerous to submit to the inoculation.
The root of the mano de sapo is usually taken fresh. There is another indispensable precaution: while undergoing this treatment it is necessary to abstain from all sexual intercourse for three days after the first inoculation, for two days after the second, and for one day after the third.
For the inoculation a large snake’s tooth, that is to say, one of the fangs, is employed, and the fangs of the most poisonous snakes, such as the rattle-snake (cuatro narices), are selected. The snake must be killed on a Friday, and the fangs extracted the same day. The same fang may serve for several years!
The inoculation is commenced on the dorsal surface of the left foot; care must be taken to avoid coming into contact with a vein. The skin is torn with the point of the fang, so that it bleeds a little, and the incision is in the shape of a square.
From the left foot the operator passes to the right wrist (anterior surface), then to the right foot (dorsal surface), and left wrist (anterior surface), always changing from one side of the body to the other.
Operations are continued on the left thigh, then on the right arm, right thigh, and left arm; in this way all the limbs are inoculated. On the body an inoculation is made in the centre of the sternum; another is made in the nape, and a final one in the centre of the forehead. The finishing touch is given with the semblance of a square incision in the tongue.
At least seven series of similar inoculations are necessary to protect a man from the spells of the serpent, and at the same time to confer upon him the faculty of curing by suction the bites of the venomous snakes that are most dreaded.
During the whole of the period in which the Indian thus submits to successive inoculations, his health shows no noteworthy derangement. He feels a slight headache and a strange inclination towards alcoholic drinks. But when the moon is at the full, then indeed, an excitement which is dangerous in another way takes possession of him. His cerebral faculties become over-excited, and he feels that his senses are deserting him; his eyes become bloodshot, and he is pursued and tormented by an irresistible impulse to bite. He has itching sensations in his gums, his mouth burns, and salivation is greatly increased. He feels that he is going to give way to the necessity to bite, and then he flees to the woods, where he bites the trees viciously, tears their bark and discharges his venom. His poisonous saliva mingles with the sap, and, surprising phenomenon, the tree withers and dies!
Woe to the man or animal who happens to be bitten by a Curado de Culebra in a fit of passion. The victim will die as quickly as if he had been bitten by a snake!
Almost all the semi-savage people of Guiana, and of the valleys of the Orinoco and the Amazons, as also the tribes of Central Africa and the races of India, possess witch-doctors, who pretend to be in possession of means to preserve themselves from snake-bites, which are just as ridiculous and infallible as the procedure described above.
The archives of a criminal anthropology contain the story of a Lyonnese gold-seeker, who had himself immunised against venom by an aboriginal native of Guiana:[91]—
“The Indian took, from a bottle which contained several of them, a tooth of the Grage (Lachesis atrox), an extremely poisonous snake, and with it made upon my instep three incisions about 3 centimetres in breadth. He allowed the wounds to bleed for a minute. I then experienced a fainting sensation, and large drops of sweat rolled from my forehead. The wounds were next rubbed with a blackish powder. I have since learnt that this powder was composed of the liver and gall of the animal, dried in the sun and pounded up with the poison-glands. The blood immediately ceased to flow. The Indian chewed some leaves of a tree mixed with this powder, and, applying his lips to the sore, injected into it as much saliva as he could, making an effort as though to inflate a balloon. This completed the operation.
“Since then I have been bitten seven times by different species of very dangerous snakes, such as the Grage, coral-snake, &c., and have never even had an attack of fever. The Galibi, Boni, and Emerillon Indians, the Bosse negroes, and all the aboriginal natives of Guiana employ the same method of procedure. They even pretend that this kind of vaccination is transmissible to their offspring, and that the hereditary immunity is maintained through several generations.”
Some years ago Mons. d’Abbadie communicated to the Académie des Sciences[92] a note from Colonel Serpa Pinto relating to another method of vaccination employed by the natives of Mozambique, which the Colonel himself consented to undergo.
“I was vaccinated,” writes Colonel Serpa Pinto, “at Inhambane (on the East Coast of Africa), among the Vatuas. These people extract the poison of a snake which is known in Portuguese as the Alcatifa (i.e., carpet), and is so called on account of the variegated colour of its skin, which resembles a carpet. I am not acquainted with the means employed in order to obtain the poison, which is mixed with vegetable substances, and forms with the latter a dark brown viscid paste.
“Two parallel incisions, 5 millimetres in length, are made in the skin, and into these is introduced the paste containing the poison. These incisions are made on the arms, near the junction of the radius and ulna with the carpal bones, on the back of the hand, on the back, on the shoulder-blades, and on the feet, near the great toes. After the operation the natives exact an oath that the vaccinated one will never kill a poisonous snake, because they say that henceforth the snake is his intimate friend, and they throw upon him an Alcatifa snake, which does not bite him.
“After undergoing this operation my whole body was swollen up for a week, and I underwent every possible kind of suffering.
“I have never been bitten by any snake, and cannot vouch for the infallibility of this remedy. The Vatuas do so, however, and they never kill a snake.
“A short time after having been vaccinated, I was stung, when in the Seychelle Islands, by a scorpion, which did me no harm. Ten years later, at the time of my journey across Africa, I was stung by another scorpion which hurt me dreadfully, and for a week I thought that I was going to die or lose my arm.”
Mystification and superstitious ideas play, as we see, a very great part in this preventive treatment, which is undergone by the natives of certain countries and snake-catchers or charmers. But it is not very surprising that, thanks to successive and repeated inoculations, a man can succeed in acquiring sufficient immunity to preserve himself from snake-bites.
In ancient times it was even pretended that it was possible for this immunity to be transmitted in certain cases by heredity, and thus we can understand how the profession of snake-charmer was hereditary in certain native families in India or Egypt.
With reference to this subject, Professor Landouzy, in his fine work on serum therapeutics, quotes a passage from “The Pharsalia” of Lucan describing, in the year 60 A.D., the customs of the Psylli, a people encountered by the army of Cato during its sojourn in Africa. This passage is so interesting that I cannot refrain from reproducing it:—
“Alone unharmed of all who till the earth
By deadly serpents, dwells the Psyllian race.
Potent as herbs their song; safe is their blood,
Nor gives admission to the poison germ
E’en when the chant has ceased. Their home itself
Placed in such venomous tract and serpent-thronged
Gained them this vantage, and a truce with death,
Else could they not have lived. Such is their trust
In purity of blood, that newly born
Each babe they prove by test of deadly asp
For foreign lineage. So the bird of Jove
Turns his new fledglings to the rising sun,
And such as gaze upon the beams of day
With eyes unwavering, for the use of heaven
He rears; but such as blink at Phœbus’ rays
Casts from the nest. Thus of unmixed descent
The 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 veritable active immunity by means of repeated inoculations of venom. We shall shortly see that the case is the same with regard to animals.
PART III.
ANTIVENOMOUS SERUM THERAPEUTICS.
CHAPTER XIII.
VACCINATION AGAINST SNAKE-VENOM—PREPARATION OF ANTIVENOMOUS SERUM—ITS PREVENTIVE PROPERTIES AS REGARDS INTOXICATION BY VENOM.
So long ago as the year 1887 it was shown by Sewall, in an important paper on “Rattlesnake-Venom,”[94] that 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 Kaufmann[95] obtained 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,[96] I arrived at the conclusion that it was possible, by means 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, that the 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 are repeated 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 properties in 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,[98] and 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 pupils or 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.
After this I was not long in pushing the vaccination of a few horses until I made them resist, in a single injection, 2 grammes of dry cobra-venom, a dose about eighty 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 succumb in 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 of sixteen months is 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.
When a horse is well vaccinated and tolerates without a reaction 2 grammes of 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 days after 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 2 grammes of 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 more grammes. 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 power in 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.
The preventive power may be very quickly tested by injecting a rabbit, in the marginal vein of the right ear for 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 by intravenous injection is 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 of their 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, and hæmorrhagin (Flexner and Noguchi), or proteolytic 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 of Colubridæ in general is characterised by the constant predominence of neurotoxin, to which it owes its extreme toxicity, which is especially intense in the case of cobra-venom. It contains no, or scarcely any hæmorrhagin; for this reason the local symptoms of poisoning by Colubrine venom are almost nil. This neurotoxin, as we have seen, shows itself very highly resistant to heat.
The venom of Viperidæ, on the contrary, especially that of Lachesis, is characterised by the almost total absence of neurotoxin, while its richness in hæmorrhagin is considerable. Consequently, heating for a few minutes at + 75° C. renders it almost entirely inactive, since hæmorrhagin is 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 the Colubridæ or Viperidæ, by determining its richness in neurotoxin resistant to heating at + 85° C.
Certain Viperine venoms, such as those of the European Vipera berus and Vipera aspis, the African Cerastes and American Crotalus contain at the same time a small proportion—varying greatly in amount according to the species—of neurotoxin, and a much larger proportion of hæmorrhagin. 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, some Colubrine venoms, such as those of Bungarus cæruleus, which are very rich in neurotoxin, 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 Australian Colubridæ (Hoplocephalus, Pseudechis) form a special group, which is richer in hæmorrhagin than are those of the Colubridæ of the Old World.[99]
On studying, in the case of these various venoms, the action in vitro and in vivo of a purely antineurotoxic antivenomous 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 less neurotoxin. It prevents hæmolysis in 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 act in vitro on those Viperine venoms 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 the neurotoxic substance is one and the same, and always neutralisable by an antineurotoxic serum like that of animals vaccinated against cobra-venom.
Neurotoxin being 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 an antineurotoxic power 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.
Antineurotoxic serum 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 to Colubrine and Viperine venoms, the neurotoxic activity of which may cause death. But it does not possess any preventive action upon the local effects of hæmorrhagin, to which the noxiousness of certain Viperine venoms—such as those of Lachesis—are almost exclusively due.
In countries in which Viperidæ are very common, we must therefore not confine ourselves to vaccinating the animals that produce serum solely against the neurotoxin of 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 of Lachesis, Vipera russellii, Crotalus, Hoplocephalus, or Pseudechis. 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 method polyvalent serums capable of preventing the local action of Viperine venoms, and of suppressing in vitro their 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 part antineurotoxins, which I have been able to do, thanks to the abundant provision of Cobra- and Bungarus-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.
CHAPTER XIV.
NEUTRALISATION OF VENOM BY ANTITOXIN.
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,[100] that the phenomenon in this case was a purely physiological one, which I considered to be proved by the fact that, if we mix in 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,[101] who 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 heating did 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 with lecithin, a combination which results in the formation of hæmolysing and non-toxic lecithides, the neurotoxin being 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 neutralising in 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 small quantity 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 gramme neutralised, it is necessary to mix with this twenty times lethal dose a dose of serum a little larger than 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 the mouse as test animal, we employ the rabbit, 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 power in vitro of each specimen of serum. But it must not be forgotten that the 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 the neurotoxic action of venoms and their hæmolytic action, 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 hæmolysing lecithide.
The knowledge of this fact enables us to determine, by means of 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 its antihæmolytic power.[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 of Colubrine venom, 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 of Lachesis or Vipera berus.
By a method calculated upon the foregoing, it is likewise possible to measure the antihæmorrhagic activity of an antivenomous serum, for the parallelism existing between the antineurotoxic and antihæmolytic actions of serums occurs again, as I have been able to establish in conjunction with Noc, between the antihæmorrhagic and antiproteolytic action of the same serums.
Now, the antiproteolytic action is easily determined by means of a series of test-tubes containing the same quantity of 20 per cent. gelatinised bouillon, 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 of toxin + antitoxin, J. Morgenroth[103] has 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 mixed in vitro with cobra-venom, remains entirely inert when brought into contact with the combination lecithin + venom, that is to say, with cobra-lecithide.
(2) That the addition of lecithin to a neutral combination of venom + antivenomous serum does not set the venom free again, and that under these conditions no lecithide is formed.
If, in a neutral mixture of cobra-hæmolysin and antitoxin we could succeed in dissociating the two constituent elements, and in then making the cobra-hæmolysin combine with the lecithin, we should have a toxin and antitoxin side by side; for the reasons indicated above, this toxin (lecithide) and antitoxin (antivenomous serum) 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 a lecithide.
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 of lecithide, 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.
CHAPTER XV.
TREATMENT OF POISONOUS SNAKE-BITES IN MAN AND ANIMALS.
OBJECTS OF THE TREATMENT: TECHNIQUE OF ANTIVENOMOUS SERUM-THERAPY.
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 chiefly efficacious against the bite of the Chalcis, Cerastes, Seps, Elaps and Dipsas.”
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 way permanganate of potash, chromic acid, chloride of gold, and the alkaline hypochlorites, especially hypochlorite of lime, may be extremely useful under many circumstances.
Permanganate of potash was recommended in 1881 by Professor de Lacerda,[104] of 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 mixed in vitro with 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 to chromic acid (1 per cent. solution), recommended by Kaufmann[105] for the bite of the common viper.
No other effect is produced by a 1 per cent. solution of chloride of gold, or the alkaline 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 is hypochlorite 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 are nil.
No better results are obtained by injections of strychnine, as recommended by Dr. Mueller, in Australia. Moreover, the statistics published by Raston Huxtable[106] positively 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 the vitality 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 2 per cent. solution of hypochlorite of lime, or with a 1 in 1,000 solution of chloride of gold. In default of hypochlorite of lime or chloride of gold, either eau 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 in making 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]).
Fig. 95.—Technique of the Injection of Antivenomous Serum beneath the Skin of the Abdomen.
If it has been impossible to apply the treatment until several hours after the bite, and if the latter has been inflicted by a poisonous snake of large size or belonging to a very dangerous species, such as the Cobra or Indian Krait, it is preferable to inject into the patient three whole doses of serum at once.
In cases in which the phenomena of serious intoxication have already appeared, and when asphyxia threatens, one must not hesitate to inject 10 or even 20 c.c. of serum directly into a vein. For such an injection it is most convenient to choose a superficial vein at the elbow or wrist, or on the back of the hand.
The introduction of serum into the veins is never dangerous if good care be taken not to allow either bubbles of air or particles of precipitated albumin to enter.
It is not advisable to repeat the injections beneath the skin or into the veins unless the general symptoms appear to become more acute.
In most cases the local pain, excitement, and attacks of cramp and nausea disappear within a few minutes after the first injection. Improvement progresses very rapidly, and by the following day the patient has recovered.
The administration of ammonia, alcohol, morphia, or ether by the mouth is entirely superfluous. These drugs, as I have already stated, may even be harmful to the patient and hinder the effects of the serum. All that should be done is to give copious hot drinks, tea or coffee, and to cover up the patient warmly in order to induce abundant perspiration.
The bitten member should not be cauterised with red hot iron or with chemical agents of any kind, since such cauterisations only lead to injuries which are too often prejudicial to the normal action of the affected organs.
Treatment of Poisonous Bites in the Case of Domestic Animals.—It often happens that dogs, horses, or cattle are bitten and succumb to the poisoning in a few hours or in two or three days. Such accidents are especially frequent among sporting dogs, even in Europe, in regions in which vipers are found.
In most cases, dogs, horses, and cattle are bitten on the nose, and such bites are immediately followed by a very painful swelling, which arouses the suspicion of the owners of the animals. It is then necessary, as soon as possible, to inject subcutaneously in the right or left flank, or at the base of the neck, one or two doses of antivenomous serum, according to the gravity of the effects observed.
The injection of the serum and the dressing of the wound should be performed as in the case of poisonous bites in human beings.
Influence of the Doses of Antivenomous Serum injected, and of the Time that has elapsed since the Venomous Bite.—I have stated above that antivenomous serum possesses a preventive and curative power of such intensity, that it is capable in a few minutes of rendering animals into which it has been injected absolutely insensible to the most strongly neurotoxic venoms, such as those of Naja or Bungarus. On the other hand, I have established the fact that, the more sensitive are the animals to intoxication by venom, the greater is the quantity of antivenomous serum necessary to immunise them passively or to cure them.
In experimenting upon mice, guinea-pigs, and rabbits, it is found that in order to preserve, let us say, a mouse of 25 grammes against inoculation with half a milligramme of venom, which is ten times the lethal dose for this little animal, it is necessary to give a preventive injection of 1 c.c. of serum; while half a cubic centimetre of the same serum is sufficient to render the dose of half a milligramme of venom innocuous, when venom and serum are mixed in vitro before being injected.
In the case of the guinea-pig, it is likewise found that the dose of serum to be injected preventively, in order to protect the animal from intoxication by ten times the lethal dose of venom, is about twice as much as the quantity of the same serum that it is sufficient to mix in vitro with venom, in order to render ten times the lethal dose of venom innocuous.
If we inject into animals first venom, in doses calculated to kill the controls of the same weight in from two to three hours, and the serum fifteen minutes afterwards, it is found that the quantity of serum that must be injected in order to prevent death is about thrice as great, as that which neutralises in vitro the dose of venom inoculated.
It is also found that the amount of curative serum that an animal intoxicated by venom must receive is inversely proportional to its weight.
The experiments upon dogs, performed at the Pasteur Institute at Lille by my collaborator C. Guérin, are highly demonstrative in this respect.[107]
A dog of 12 kilogrammes, inoculated with 9 milligrammes of venom (a dose lethal to controls of the same weight in from five to seven hours), is completely cured on receiving, two hours after inoculation with the poison, 10 c.c. of serum.
When the treatment does not take place until three hours after the injection of the venom, it is necessary to inject 20 cc. of serum in order to prevent the animal from dying. With a longer delay than this, death is inevitable, since the bulbar centres are already affected, and paralysis of the respiratory muscles commences to appear.
These facts show that:—
(1) The more sensitive animals are to venom, the greater is the quantity of serum necessary in order to prevent their intoxication by a given dose of venom.
(2) For a given species of animal and a given dose of venom, the longer the delay in applying the remedy, the greater is the quantity of serum that must be injected in order to arrest the poisoning.
It will be understood from what has been already stated, that a man weighing 60 kilogrammes, if bitten by a snake which injects, let us say, what would amount to 20 milligrammes of venom if collected in the dry state (the mean quantity that a Naja is able to inoculate in a single bite), would only require, in order to escape death, to receive the quantity of antivenomous serum sufficient to neutralise the portion of venom in excess of the amount that he could tolerate without dying.
Let us suppose, for the sake of example, that the man of 60 kilogrammes can withstand intoxication by 14 milligrammes of Naja-venom. It follows that, in the case with which we are dealing, we must inject sufficient serum to neutralise 20-14 (=6) milligrammes of venom; that is to say, the injection of serum being made immediately after the bite, 6 c.c., if the serum employed neutralises in vitro 1 milligramme of venom per cubic centimetre.
Of course, if the serum is more powerful, less of it will be necessary, while more will be required if the remedy is applied later, or if the quantity of venom inoculated by the snake is supposed to have been greater.
For this reason, in practice, but very little serum is usually necessary in order to augment the natural resistance of a man of average weight or of a large animal; it is sufficient in most cases to give an injection of 10 or 20 c.c. in order to cure human beings who have been bitten. The clinical proof of this is, moreover, to be found in the cases, already very numerous, that have been published in the course of the last few years in the scientific journals of all countries. I have gathered together a few of these in the concluding pages of this book, and I would beg the reader to be good enough to refer to them.