The physiological effects of the various venoms are very different from those that we have just described, when these toxic substances are introduced into the organism otherwise than subcutaneously.
Their direct penetration into the blood-stream, whether by the bite of the snake itself or by experimental intravenous injection, always produces immediate results. With the venoms ofViperidæ, coagulation of the blood and, consequently, death are almost instantaneous. With the venoms ofColubridæ, which, on the contrary, destroy the coagulability of the blood, the toxic effects are less rapid, but after the lapse of only a few minutes asphyxia ensues and the death-struggle is very short.
Absorption by the serous membranes is slower, but is nevertheless effected much more quickly than when it takes place in the subcutaneous cellular tissue. When cobra-venom is injected into the peritoneal cavity of a rabbit or a guinea-pig, the local effects upon the serous membrane are almostnil. No leucocytic exudation is observed; death supervenes before this has had time to take place. The venoms ofViperidæ, on the contrary, produce, directly after their introduction into the peritoneum, an enormous afflux of sanguinolent serosity; the capillary vessels of the serous membrane, immediately becoming distended, allow the blood to filter through their walls, and the animal succumbsafter a few minutes, or a few hours, according to the dose injected, with the peritoneum full of blood.
When deposited upon the mucous membranes of the eye, vagina, or urethra, all venoms, those ofColubridælike those ofViperidæ—but the latter with greater intensity—cause very acute inflammation, comparable to that produced by jequirity; the capillaries become distended, allow leucocytes to exudeen masse, and, as for instance upon the eye of the rabbit, a purulent ophthalmia soon establishes itself.
Certain species ofSepedon(Colubridæ), common on the West Coast of Africa, especially in Senegambia and in the hinterland of Dahomey, and to which the nameSpitting Snakeshas been given, possess the faculty of projecting little drops of venom to a distance by forcibly expelling the air from their lungs, and the natives assert that this venom, when it happens to come into contact with the eyes, causes blindness. This is true to a certain extent, in so far as it produces attacks of purulent ophthalmia which are often serious; but these attacks, like those provoked experimentally in animals, can be cured in a few days when properly treated.
When absorbed by thedigestivetract, the venoms ofColubridæoften produce no ill-effects. It is otherwise with those ofViperidæ. The venom ofLachesis, for example, if administered in sufficient doses, sets up acute inflammation of the gastric mucous membrane, and the animals speedily succumb with attacks of gastro-intestinal hæmorrhage, even before it has been possible for the toxic effects upon the nerve-cells to become apparent.
These facts explain the contradictions that are to be found in the works of different investigators upon this subject. It is affirmed by some writers that venom can be swallowed without danger, and they even advise the sucking of venomous wounds in order to hinder its absorption. Others, including Sir Joseph Fayrer, Richards, and Weir Mitchell, have killed pigeons and fowls by making them ingest venom ofVipera russellii, orCrotalus.C. J. Martin, in experimenting upon rats with the venom ofPseudechis(Colubridæ), has succeeded in keeping these animals alive for a whole week by providing them every day with a ration of bread and milk mixed with a dose of venom one hundred times greater than the lethal dose for a subcutaneous injection. This innocuousness of the venoms ofColubridæ, which I have frequently been able to establish by causing them to be ingested by different animals, is explained by the fact that the pancreatic juice and the ptyalin of the saliva very rapidly modify the proteic substances to which these venoms owe their toxicity, so that this disappears. No trace of them is found in the fæces.
The glandular secretions of persons bitten by venomous snakes, and those of animals inoculated with doses of venom calculated to kill only after a few hours, are not infrequently found to be toxic. In the case of the urine in particular this has been shown to be so.
Observations have also been recorded by C. Francis18and Sir Joseph Fayrer with reference to the passage of venom through the mammary gland. In the year 1893 a poor Mussulman woman died at Madras from the bite of a Cobra. She was nursing her child at the time, and the latter succumbed in its turn a few hours later, with all the symptoms of poisoning, although it had not itself been bitten, and had been suckled by its mother only once since the bite.
Thehistological lesionsproduced by snake poisoning have been particularly well studied by Hindale,19Karlinski,20Nowak,21Louis Vaillant-Hovius,22and Zeliony.23
Whether we are dealing with the venoms ofViperidæorColubridæ, the anatomo-pathological processes are alike, and the changes produced are more or less profound, according to the degree or the slowness of the intoxication.
The liver is more affected than any other organ. In cases in which death has quickly followed the injection of the venom, the protoplasm of the cells is merely cloudy, or granular, and the granulations readily take a stain in their periphery, though the interior remains uncoloured. If, on the contrary, the animal has survived for some hours, the protoplasm becomes condensed in certain parts of the cell, leaving vacuoles, the limits of which are not well defined. A portion of the cellular protoplasm is necrosed and destroyed. In these cases the nuclei have already undergone a change; although their contours may be well defined, we discover in their interior only a very little chromatin in the form of small granulations, and the nuclear fluid takes a feeble stain with basic colours, since it contains a little chromatin in solution.
When the protoplasm of the hepatic cells has suffered more pronounced lesions, the changes in the nuclei are also more marked; the quantity of nuclear chromatin diminishes and slowly loses its property of taking stains, in proportion as the protoplasm of the hepatic cells undergoes necrosis; finally, in the hepatic cell, there remains nothing more than a small quantity of granular protoplasm without a nucleus (Nowak).
In certain cases we find extensive areas of fatty degeneration, or small foci in which the hepatic tissue is absolutely destroyed. In the case of the dog it may even happen that the microscopic structure of the parenchyma has entirely disappeared. The arrangement of the hepatic cells in lobules can no longer be distinguished; the trabeculæ are ruptured and broken asunder, and we find nothing more than a confused agglomeration of cells floating in the extravasated blood.
In animals which have lived for a long time after being poisoned, lesions of the bile-ducts are also found. The epithelial cells have undergone fatty degeneration, or else, in the case of small animals, the ducts appear infiltrated with small mononuclear cells, which penetrate between the epithelial cells of the canaliculi. Sometimes also the latter cells are distended, and enclose large vacuoles.
Venom thus produces in the liver lesions offatty degeneration, ornecrosis, and an infiltration of the bile-ducts by lymphatic cells.
The changes in the kidney are also very extensive. The three portions of the glomerulus often exhibit lesions; the vessels of the tuft show ectasia; their walls are sometimes ruptured, and the blood is extravasated into the capsular cavity. The latter is filled with a granular exudation, which varies in amount with the slowness of the intoxication. The epithelial lining ofBowman’s capsuleis swollen; the nucleus stains badly (Vaillant-Hovius).
In thetubuli contortithe lesions in the cells greatly resemble those seen in the liver. Granulations and vacuoles appear, and the nucleus becomes diffuse. The lumens of the tubules are filled with necrosed cells, and thebranches of Henleare found to be similarly obliterated.
In thestraight tubesand in thecollecting tubesthe epithelium is sometimes detached in its entirety. Some of these canals are obliterated by granular cylinders or by accumulations of epithelial cells.
The vessels met with in the parenchyma of the kidney are always greatly distended, and sometimes they are torn, whence there results the formation of small foci of interstitial hæmorrhage. In many cases the extravasated blood also destroys the parenchyma.
In the spleen, Nowak merely found a little fatty degeneration, and only in cases in which the lesions in the liver and kidneyswere very far advanced. The same applies to the muscular fibres of the heart. This organ exhibits, above all, hæmorrhagic infiltrations in its peripheral portion, rarely in its substance.
The lungs are the seat of more important lesions. We find in them a multitude of little infarcts. Around these the capillary vessels are extremely dilated, and the pulmonary vesicles have become very small.
All these lesions of the visceral organs strangely resemble those observed in the case of individuals who have died fromyellow fever. This observation has been made by several scientists, among others by Sanarelli, and it is this perhaps that has suggested to some (Dyer, of St. Louis, R. Bettencourt, of São-Paulo24) the idea of treating—without much success, however—yellow fever by the antitoxin of venom.
The changes in thestriated musclesin places at which venom has been injected do not present any specific character. The muscular fibres already become necrosed half an hour after the injection; the diseased tissue becomes permeated with an albuminous mass rich in fibrin, and the blood is extravasated. A few hours later we observe, between the bundles of degenerate muscle fibres, polymorphous leucocytes. The number of these latter constantly increases, and attains its maximum after one or two days. The muscular nuclei become distorted, appear long or angular, and assume the aspect of myoblasts (sarcoblastic muscle cells). In the protoplasm of the myoblasts we frequently find particles of broken-down muscle, and globules of fat.
All these changes resemble those observed as the result of the action of a host of other muscle poisons, especially the irritant or caustic chemical substances.
It is extremely difficult to determine with any degree of precision the nature of the lesions produced by venoms in thenervous system. The intensity of these lesions depends in the first place upon the length of time that has elapsed between the introduction of the venom into the organism and death. It depends, secondarily, in a large measure, upon the origin of the venom. That of theViperidæacts almost exclusively upon the blood by coagulation, and exhibits only a very slight degree of toxicity as regards the nerve-cell. That of theColubridæ, on the contrary, produces manifest changes in the chromatic substance. Nissl’s bodies are completely disintegrated, and transformed into a granular mass. In the majority of the stichochromes neither the form of the bodies nor even the reticulum is distinguishable. The nuclei are opaque, the nucleoli swollen and broken up. The dendrites often become irregular and contracted (Ewing and Bailey,25G. Lamb26).
It was found by Bailey that the majority of the cells of the anterior cornua of the medulla are normal, but that a small number of them exhibit indications of acute granular degeneration; a few cells were found to have lost almost all their chromatic substance.
From the physiological point of view it is perfectly clear that Cobra-venom especially affects the bulbar centres, and particularly the nuclei of origin of the pneumogastric nerve. We observe in the first instance the gradual suppression of the functions vested in the nerve-cells that are found in connection with the vagus nerve, the spinal accessory, and the hypoglossal. Later on the excitability of the nerve-endings in the muscles is found to have been destroyed, and this action presents great similarity to that of curare.
The venoms ofViperidæ, when injected in very weak doses, exercise a paralysing action upon the reflex excitability of themedulla. But it is open to question whether these effects are not exclusively due to the lesions of the blood, which are here all-predominant; for no histological modification is observed in the cells of the central nervous system.
I have made a number of experiments with a view to discovering whether the cerebral, bulbar, or medullary substance of animals susceptible to the action of Cobra-venom (rabbit, guinea-pig, fowl) possesses the property offixingthis venom as it fixes the toxin of tetanus (Wassermann and Takaki). I found that, on pounding up a little of the pulp of the cerebral hemispheres or bulb with doses of venom lethal in two hours for the control animals, the injection of the mixture, well washed and centrifuged in order to free it from all excess of non-fixed venom, always caused death, but with a retardation of from four to tenhours. We see, therefore, that partial fixation of the venom upon the nervous elements really takes place, but we cannot conclude from this that these elements exercise an antitoxic function, any more than in the case of tetanus, for animals that receive cerebral emulsions in one thigh and the dose of venom lethal in two hours in the other thigh, succumb at the same time as the controls.
Major Rogers has made similar experiments with the venom ofEnhydrina(Hydrophiidæ), and has obtained the same result on employing the cerebral hemispheres of the pigeon.27
Flexner and Noguchi,28on their part, have compared, by aid of the method of intra-cerebral injections, the toxicity of the venom ofCrotaluswith that of the venom of theCobra. On employing Cobra-venom heated to 75° C., they found that the convulsive and paralytic effects were immediate, contrary to what takes place after subcutaneous or intraperitoneal injections, but that the dose of venom necessary to produce death was the same(0·1 milligramme for the guinea-pig) as when the injection is made in the peritoneum or beneath the skin.
With the venom ofCrotalusheated for half an hour at 75° C., which contains but very littleneurotoxinand has lost all its hæmorrhagic properties, 0·5 milligramme introduced directly into the brain of the guinea-pig only produces transitory and non-lethal effects; while, if fresh venom be employed, 0·05 milligramme is sufficient to cause death in three hours, with severe hæmorrhagic lesions. Now this dose is twenty times smaller than the minimal lethal dose for a subcutaneous injection.
It is evident that the harmful matter, in the particular case ofCrotalus-venom, is not theneurotoxin, but an altogether different substance, termed by Flexner and Noguchihæmorrhagin, which acts upon the elements of the blood and upon the endothelium of the blood-vessels.
We shall meet with this substance again in almost allViperinevenoms, and shall study it further on.
On making an autopsy of an animal which has succumbed to intoxication by snake-venom, we find that the blood in the heart and large vessels is sometimes coagulated into a mass, sometimes entirely fluid, and that, in certain cases, it is as black as prune-juice, while in others it is of a fine transparent red colour.
These differences in the effects of venom upon the blood are due to the fact that the various venoms contain in variable proportions, besides theneurotoxicsubstance which represents the true venomoustoxin, other substances which act, some upon the plasmasia or fibrin-ferment, or upon the fibrin, others upon the red corpuscles, others on the leucocytes, and others again on the endothelium of the blood-vessels.
It was observed long ago by Fontana29that after viper-bites the blood remains fluid, and Brainard30on the contrary, pointed out that, in the case of animals that succumb very rapidly after having been bitten by aCrotalus, the blood was always found coagulated into a mass, while, when a certain interval of time had elapsed since the bite, it remained fluid. Weir Mitchell31explained these differences by the hypothesis that, in cases of rapid death, the blood had not had time to become modified by the venom.
Later on it was found by Sir Joseph Fayrer, and subsequently by Halford,32in Melbourne, C. J. Martin,33in Sydney, G. Lamb,34in Bombay, and recently by Noc, in my laboratory, that the venoms ofColubridæ, especially those ofNaja tripudiansandAustralianspecies of this family, always leave the blood fluid after death, while the venoms ofViperidæ, on the contrary, are usually coagulant.
On the other hand, it was observed by Phisalix,35and at an earlier date by Mosso, of Turin, that the venom ofVipera beruscauses the blood of the dog to lose its coagulability, while, on the contrary, the same venom is actively coagulant as regards the blood of the rabbit.
How are these differences of action to be explained? It was found by Delezenne,36who made an excellent study of the phenomena following the injection of peptone, extracts of organs, and other anticoagulant substances into the organism, that those of these substances that render the blood non-coagulable always dissolve the leucocytes, and thus set at liberty two antagonistic bodies which they contain. One of these substances is coagulant and is found retained by the liver, while the other remains in solution in the plasma, and keeps the blood fluid after issuing from the vessels.
Now, certain extracts of organs, ricin, abrin and certain venoms in weak doses, retard coagulation, while in large doses, on the contrary, they produce partial or general intravascular clotting.
It is believed by Delezenne that the explanation of thisphenomenon may be that the doses, which are weak but sufficient to produce the disintegration of the leucocytes, injure the red corpuscle in only a slight degree, while the stronger doses are equally destructive to the two kinds of blood corpuscles.
It follows that we must understand that there are two phases in the action of venoms: onenegative, when the dose absorbed does not injure the leucocytes; the otherpositive, when the leucocytes are destroyed.
If the blood of the dog remains non-coagulable when mixed with doses of venom which, on the contrary, are actively coagulant for the blood of the rabbit, the reason would be that the leucocytes of these animals are not equally resistant to venom.
This conception, however, does not conform to the facts that I have myself observed. I have always found that viper-venom, mixed with citrate- or oxalate-plasma of the dog, rabbit, or horse, coagulates these various plasmas when the venom is in weak doses, while with strong doses coagulation is not produced. To be quite accurate, it should be stated that the quantity of venom necessary to render the plasma of the dog, or of the horse, non-coagulable is less than that which must be employed in the case of the plasma of the rabbit.
I have caused Noc to take up anew the study of this question in my laboratory, with venoms of nine different origins, and I here give arésuméof the results of his researches.37
The venoms ofViperidæstudied range themselves as follows according to their coagulant power:—
The venoms ofAncistrodon contortrixandA. piscivorus(Crotalinæ) proved entirely inactive.
NoColubrinevenom exhibited coagulant power, whatever the dose employed.
There is, therefore, a very decided difference between venoms of divers origins as regards their effects upon the coagulation of the blood.
Noc has determined more especially the coagulant action of the venom ofLachesis lanceolatus(Fer-de-lance of Martinique) upon 1 per cent. citrate-plasmas, 1 per cent. oxalate-plasmas, 4 per cent. chloridate-plasmas, and upon blood rendered non-coagulable by extract of leeches’ heads. He found that, while weak doses of venom (1 milligramme per cubic centimetre of horse- or rabbit-plasma) produce coagulation in a few minutes in the citrate-plasmas, chloridate-plasmas, or those treated with extract of leeches, the doses of the same venom greater than 4 milligrammes on the contrary suppress the coagulability of these plasmas, even when there be added to them doses of chloride of calcium (for the citrate- and oxalate-plasmas), or of distilled water (for the chloridate-plasma), or of fibrin-ferment (for the plasma treated with leech-extract) sufficient to cause rapid coagulation in the control tubes that do not contain venom.
Noc also observed that the venom of the same species of snake (Lachesis lanceolatus), when heated to 75° C., entirely loses its coagulant properties; and that, with a temperature of 58° C., its coagulant power already commences to diminish. When heated for thirty minutes at a temperature of 65° C., a dose of 1 milligramme does not coagulate more than 1 c.c. of citrate-plasma in one hour. G. Lamb has likewise found that the venom ofVipera russelliiloses its coagulant power when heated to 75° C.
The coagulant substance in these venoms is precipitable by alcohol at the same time as theneurotoxinand other active substances. The precipitate, when dissolved again in physiological water, preserves all the properties of the original solution.
Antivenomousanticolubrineserum, that is to say, that furnished by horses vaccinated against the venoms of theCobraand theKrait, does not prevent coagulation by coagulant venoms. This need not surprise us, since the coagulant substances in venoms are destroyed by heating, and the animals vaccinated in order to obtain antitoxic serum are usually inoculated exclusively with heated venoms.
It is easy, however, to obtain active serums specific against the coagulant venoms; it is sufficient to treat these animals by inoculation with progressively increasing doses of the same venoms unheated. I have had no difficulty in achieving this result with small laboratory animals (guinea-pigs and rabbits) and also with the horse, but I have never had at my disposal a sufficient amount of the venoms ofLachesisorVipera russelliito undertake with them the regular acquisition of large quantities of horse-serum, at onceantineurotoxicandanticoagulant. The preparation of such a serum, nevertheless, presents much interest for certain countries, such as Burma, where the Daboia (Vipera russellii) is almost as common as the Cobra, and Brazil, where nearly all the casualties due to venomous snakes are produced byLachesis.38
Contrary to what is observed with the venoms ofViperidæin general, all the venoms ofColubridæand, as exceptions to the rule, the venoms of some North AmericanCrotalinæ(Ancistrodon contortrixandA. piscivorus) suppress the coagulability of the bloodin vivoandin vitro. It is, however, important to observe that,in vivo, the blood remains fluid after death only if the dose of venom absorbed has been sufficient.In vitrothis phenomenon is easier to study, and has been the subject of several important memoirs.
Halford,39Sir Joseph Fayrer,40C. J. Martin,41Delezenne,42Phisalix,43and lastly Noc,44have shown that the venoms ofColubridæexert a manifestly anticoagulant action upon citrate-, chloridate-, or oxalate-plasmas, and also upon blood mixed with venom on issuing from the vessels.
On adding 1 milligramme ofCobra-,Bungarus-, AustralianPseudechis-, orAncistrodon-venom to 1 c.c. of citrate-, oxalate-, or chloridate-plasma, and supplementing the mixture, after varying periods of contact, with a quantity of chloride of calcium (for the citrate- or oxalate-plasmas), or distilled water (for the saline plasma) sufficient to produce coagulation in a few minutes in the control tubes without venom, we find that coagulation no longer takes place after one hour in the tubes containingCobra- orBungarus-venom, and after ten minutes in those that contain the venom ofAncistrodon.
In doses less than 1 milligramme for 1 c.c. of plasma, these venoms by themselves never produce coagulation as do those ofLachesisorVipera russellii. They are thus sharply differentiated in this respect.
If fresh blood issuing from the arteries of an animal be received in a vessel containing a sufficient quantity ofColubrine-venom (that of the Cobra for example), and steps be immediately taken to ensure the perfect mixture of the venom and the blood, we find that the latter has entirely lost its coagulability, just as though it had been mixed with peptone or extract of leeches’ heads.
Again, if a mixture be madein vitroof coagulant venoms, suchas that of theLachesis, with anticoagulant venoms such as that of theCobraor ofAncistrodon, it is found that these mixtures, when properly effected, become neutral, so that the respective effects of the component venoms are entirely destroyed. Assuming, for instance, that 1 milligramme ofLachesis-venom coagulates in two minutes 1 c.c. of 1 per cent. citrate rabbit-plasma, if we add to the plasma firstly 1 milligramme ofAncistrodon-, or 1 milligramme ofCobra-venom, and then 1 milligramme ofLachesis-venom, the plasma remains non-coagulated, yet coagulates perfectly on the subsequent addition of 1 c.c. of a ½ per cent. solution of chloride of calcium.
There is, therefore, a real antagonism between the actively coagulant substance contained in certainViperinevenoms and the anticoagulant substance comprised in the venoms of certain otherViperidæ(Ancistrodon), belonging to the subfamilyCrotalinæ, and in those of all theColubridæ.
The conclusion to be deduced from the foregoing facts is that the venoms ofColubridæand those of certainViperidæare decidedlyanticoagulant, while the majority of the venoms ofViperidæ, on the contrary, possess strongcoagulantproperties, even when mixed with blood in infinitesimal doses.
The question therefore arises why thesecoagulantViperinevenoms suppress the coagulability of the blood when mixed with itin vitroin strong doses (for example, in doses beginning from 4 milligrammes ofLachesis-venom, or 7 milligrammes of the venom ofVipera russelliifor 1 c.c. of 1 per cent. citrate rabbit-plasma).
The explanation of this apparently contradictory phenomenon is furnished by the intense proteolysis that theseViperinevenoms exert upon fibrin, in solution or coagulated. This proteolysis actually manifests itself with weak coagulant doses, for the compact clots formed at the outset soon become soft and then dissolve, like a cube of egg-albumen in an experiment in artificial digestion by trypsin. We shall revert to the subject later on.
The anticoagulant action of the venoms ofColubridæand ofAncistrodonupon the blood appears to take effect in the first place upon the fibrin-ferment, and afterwards upon the fibrin by proteolysis. The action on the fibrin-ferment seems manifest when we experiment with anticoagulant venoms which are feebly proteolytic, like the venom of theCobra.
I have already stated that a mixture of fresh blood with a sufficient dose ofCobra-venom is non-coagulable, as though the blood on issuing from the animal had been mixed with peptone or leech-extract. But, while blood when peptonised or mixed with leech-extract coagulates readily on the subsequent addition of fibrin-ferment, blood mixed with venom remains positively non-coagulable. It is the same with citrate- or oxalate-plasmas, which no longer coagulate when chloride of calcium is added to them, and with 4 per cent. saline plasma on the addition of distilled water.
The anticoagulant substance in the venoms ofColubridæandAncistrodonis precipitable by alcohol, like the coagulant substance in the venoms ofViperidæand like theneurotoxins, from which it is difficult to separate them. The separation can nevertheless be effected by the aid of heat, if we make use of certain venoms that are particularly resistant to high temperatures, such as those of theCobraor theKrait. These latter venoms, when heated for one hour at 70° C., cease to be anticoagulant, andpreserve their toxicity unimpaired. It is, however, impossible to suppress the toxicity without at the same time destroying the anticoagulant substance.
Antivenomous serumcompletely protects citrate- or chloridate-plasmas against the anticoagulant action of venoms. It is sufficient to mix ½ c.c. of 4 per cent. saline antivenomous serum with 1 c.c. of 4 per cent. saline plasma to ensure that the subsequent addition of 1 milligramme ofCobra-venom to this mixture remains without effect upon the coagulability of the latter. If,after a contact of two hours or more, 2 c.c. of distilled water be added, coagulation is produced just as in saline plasma without venom.
(1)Hæmolysis.—The hæmolytic properties of venoms, that is to say, their faculty of dissolving the red corpuscles, have been the subject of very important researches on the part of a number of investigators during the last few years (W. Stephens,45Flexner and Noguchi,46Calmette,47Phisalix,48Preston Kyes and Hans Sachs,49Noc50).
The different venoms are all hæmolytic, but in very variable doses. It is possible to make a very precise comparative study of them from this special point of view by taking as a base for each venom, as was done by Noc, the unital dose of 1 milligramme (or one-tenth of a cubic centimetre of a 1 per cent. solution freshly prepared and not filtered, the filtration through porcelain retaining an appreciable part of the active substance), and noting the time strictly necessary for this dose of 1 milligramme to dissolve completely,in vitro, 1 c.c. of a 5 per cent. dilution of red corpuscles of the horse in physiological saline solution.
It is very important, before allowing the venom to act on the red corpuscles, to first wash the latter by means of several successive centrifugings with 8 per 1,000 physiological saline solution.
It is also better to choose the corpuscles of the horse in preference to those of other species of animals, since they exhibita nearly constant mean sensitivity. The corpuscles of the ox, goat, sheep, and rabbit are less sensitive. Those of man, the guinea-pig, and the rat, on the contrary, are more so.
On experimenting withwashedcorpuscles, it is found that venom alone is incapable of dissolving them. In order that dissolution may take place, we are obliged to add to the mixture either a small quantity of normal horse-serum, preferablyheated, and, consequently, deprived of alexin (Calmette), or ½ c.c. of a 1 in 10,000 solution oflecithinin physiological saline water (P. Kyes).
Venom, therefore, is capable of hæmolysing red corpuscles only when it isquickened, either by heated normal serum, or by lecithin. The solution of lecithin employed for this purpose should be prepared by dissolving 1 gramme of lecithin in 100 grammes of pure methylic alcohol. Taking 1 c.c. of this dilution we add it to 9 c.c. of 8 in 1,000 saline solution, and make a second dilution of 1 c.c. of the foregoing mixture in 9 c.c. of saline water. This latter dilution of 1 in 10,000 is utilised as the reagent.
Let us now see how the serum or lecithin acts. It has been shown by P. Kyes that with either of these substances the mechanism of the hæmolytic action is the same, for the serum quickens the venom only through the agency of the free lecithin it contains. The lecithin takes part in the reaction by combining with the venom to form a hæmolysinglecithidemore resistant to heat than its two components, for it may be heated for several hours at a temperature of 100° C., without the loss of any of its properties.
When venom is brought into contact with certain kinds of highly sensitive red corpuscles, those of the rat for example, these corpuscles, although washed and freed from serum, may undergo hæmolysis. This result is due to the fact that these corpuscles contain sufficient quantities of lecithin, which becomes liberated from their protoplasm and, uniting with the venom, constitutes the activelecithide.
It was already known that lecithin is capable of combining with various albuminoid matters and with sugars to formlecithides.We must not, therefore, be surprised to find that such a combination may take place with the proteic substances in venom. The combination in this case is a truly chemical one. Lecithin in its natural state, or that which is normally found in serums which quicken venom, such as horse-serum, even when heated to 65° C., therefore plays the part ofcomplementaccording to Ehrlich’s theory, or that ofalexinaccording to the theory of Bordet, while venom itself would be anamboceptororsensitiser.
This is not, however, the way in which the phenomenon should be understood, for it is impossible to admit the identification of heated serum or lecithin with the complementary substances or alexins, seeing that the essential characteristic of the latter is that they are intolerant of heat and become entirely inactive on being raised to a temperature of 58° C., or even by simply being kept for a few days exposed to the air and light. We must therefore suppose, with P. Kyes and H. Sachs, that the red corpuscles themselves contain substances capable of playing the part of complements (endo-complements), and that it is with these that the venom combines when quickened by the presence of lecithin or heated serum, the latter only acting because it contains free lecithin.
All substances that contain lecithin, such as bile, hot milk, or cephalin, are capable of exerting the same quickening action, but do not themselves possess any inherent hæmolytic power.
Cholesterin, on the contrary, represents a kind of antidote to lecithin, as also to normal serums. It prevents hæmolysis of the red corpuscles in a mixture of washed corpuscles and venom, yet it does not in any way modify the properties of true alexins or complements.
Moreover, no correlation exists betweenlecithidesand theneurotoxinin venoms. The combination lecithin + venom possesseshæmolyticaction, but is in no wayneurotoxic. Conversely, venom can be freed from its groups of molecules combinable with lecithin, and remainneurotoxic.
Lecithideis insoluble in ether and acetone, but soluble inchloroform, alcohol, toluene, and water. Its properties are therefore entirely distinct from those of its two components. It precipitates slowly from its aqueous solutions, without losing its hæmolytic power; it does not showbiuret-reaction; it dissolves with equal readiness the red corpuscles of all species of animals, and its effects, like those of venom, are impeded by cholesterin.
Kyes has succeeded in obtaining lecithides with all the hæmolytic venoms that he was able to study: thus he has prepared lecithides fromLachesis lanceolatus,Naja haje,Bungarus,Lachesis flavoviridis, andCrotalus. It is therefore probable that thelecithinophilegroup exists in all venoms, even when these differ as regards their other properties.
A wide range of difference is exhibited by the various venoms, as regards their hæmolysing power in the presence of normal heated serum or lecithin. The venom ofNajaand that ofBungarusare the most active. The action of the venoms ofViperidæ, and especially of those ofCrotalus, is very weak. For example, while 1 milligramme ofCobra-venom dissolves in from five to ten minutes 1 c.c. of a 5 per cent. dilution of red corpuscles in the presence of lecithin or normal heated serum, the same dose of the venom ofVipera russelliitakes thirty minutes to effect the dissolution, and the venom ofLachesis lanceolatustakes three hours.
P. Kyes and H. Sachs have discovered the apparently paradoxical fact that, if to the red corpuscles of certain species of animalsCobra-venom be added in increasing doses, hæmolysis augments up to a certain point, beyond which the destruction of the corpuscles shows progressive diminution. In a large doseCobra-venom no longer produces any effect upon the corpuscles of the horse, for example, even when the venom is added in presence of a great excess of lecithin or heated serum. It would seem, then, that, according to the theory of Ehrlich, under the influence of an exaggerated amount of venom-amboceptor there is produced a deviation on the part of the complement (serum orlecithin), and that the latter, instead of fixing itself upon the corpuscles, becomes united with the surplus fraction of the amboceptors, which has remained free in the liquid.
Noguchi,51resuming the study of this extremely curious action of strong doses of venom, observed that the red corpuscles of certain species of animals (such as the horse for example), when previously washed and held in suspension in a physiological solution of sea-salt containing 4 per cent. ofCobra-venom, acquire a considerable augmentation of resisting power with regard to various physical and chemical agents. In consequence of this they are no longer hæmolysed by distilled water, ether, or saponin.
Nevertheless, acids or alkalies, except ammonia, destroy corpuscles treated with venom more easily than those in their normal condition.
If corpuscles, previously treated with a strong dose of venom, are subjected to repeated washings in physiological saline solution, the special resistance acquired by them in the presence of the venom disappears; they even become more sensitive to the action of destructive agents, such as water, ether, or saponin.
The principle contained in venom, to which must be attributed the protective action, is not destroyed by heating to 95° C., although at this temperatureCobra-venom becomes partially coagulated. Moreover, the protective substance is contained in the coagulum, while thehæmolysinremains entirely in the filtrate. The agglutinin of venom, on the other hand, is destroyed at a temperature of 75° C. The protective substance, therefore, can be identified neither with the hæmolysin nor with the agglutinin.
It follows that it is impossible to accept the hypothesis of the “deviation of the complement” suggested by Kyes and Sachs to explain the innocuousness of strong doses of venom. Besides, it would be difficult to reconcile this hypothesis with the fact, observed by Noguchi, that venom in a strong dose protects corpuscles,not only against the action of lecithin (complement), but also against distilled water, ether, &c.
Noguchi, seeking more thoroughly to elucidate the mechanism of this protective action, finds thatCobra-venom forms a precipitate with blood-serum, when the latter is relatively poor in salts or when it is dilated with water. It likewise forms a precipitate with the aqueous extract of red corpuscles, and precipitates the globulins, hæmoglobin, or globin of the corpuscle, when treated separately. The precipitates are insoluble in water, but dissolve with the assistance of a small quantity of acid or alkali, and also in a great excess of saline solution.
Noguchi supposes that red corpuscles, when treated with strong solutions of venom, are protected against destructive agents on account of the formation by the venom and certain constituents of the corpuscle (chiefly hæmoglobin) of a compound insoluble in water. When this compound is removed by repeated washings in physiological solution, the corpuscles can easily be hæmolysed afresh by the ordinary destructive agents. Venom, none the less, exerts a noxious influence upon the corpuscles in all cases; but when strong solutions are employed, this effect is masked by the protective action.
All kinds of red blood corpuscles are not equally sensitive to the protective action of strong doses of venom. In this respect all degrees are observed in the action of venom. Thus the corpuscles of the dog are not protected at all byCobra-venom. But it is interesting to observe that this venom in no way precipitates either the aqueous extract of dog’s corpuscles, or the hæmoglobin, or the globin of this animal.
The venom ofCrotalusand that ofAncistrodonlikewise possess protective power, which is, however, less marked than in the case ofCobra-venom.
Noguchi finally points out that corpuscles treated with venom are not hæmolysed by fluorescent substances such as eosin. They are also refractory to the hæmolysing action of tetanolysin.
The resistance of the hæmolysins of venom to heat (which, according to Morgenroth, may extend to heating for thirty minutes at a temperature of 100° C.) explains how it is that the serum of horses immunised by means of venoms heated to 72° C. is distinctly antihæmolysing, and capable of perfectly protecting the red corpusclesin vitroandin vivo.
I have been able to prove that theantineurotoxicproperty of antitoxic serums with regard to the venoms ofColubridæis pretty much on a par with their antihæmolysing property, so that it is possible to measurein vitrothe antitoxic activity of a serum by establishing the degree of its antihæmolysing activity. Thus we see that a serum, which is antitoxic and antihæmolytic with respect to the venom ofNaja, is likewise antihæmolytic as regards the otherColubrine-venoms, and even certain venoms ofViperidæ. Here we have a very important fact, for it enables us to measurein vitrothe activity of antivenomous serums.
(2)Precipitins of Venoms.—The serum of rabbits treated with increasing doses ofCobra-venom precipitates the latter in more or less concentrated solution. It has no effect as regards other venoms. On the other hand, the serum of a strongly immunised horse, the antivenomous power of which was pretty considerable, gave no precipitate withCobra-venom; the formation of precipitate is therefore entirely independent of that of antitoxins (G. Lamb).52
(3)Agglutinins of Venoms.—Besides their hæmolytic action, it is easy to observe that certain venoms, especially those ofViperidæ, agglutinate the red corpuscles, and that the substance that produces this agglutination is different from the hæmolysing substance; for it acts rapidly at a temperature of O° C., at which hæmolysin manifests its effects only with extreme slowness. Heating to 75° C. destroys this agglutinant property (Flexner and Noguchi).
The white corpuscles themselves do not escape the action of venom. It is possiblein vitroto prove this action upon leucocytic exudations obtained,e.g., by injecting sterilised cultures ofBacillus megatheriuminto the pleura or peritoneum of the rabbit. After a few hours this exudation is removed by means of capillary tubes, and, on mixing these with weak doses of venom, we see, in the course of a microscopic examination, that the large mononuclear cells are the first to be dissolved, then the polynuclears, and lastly the lymphocytes. The leucolysis is much more intense and more rapid withCobra-venom than with that ofCrotalus(Flexner and Noguchi, Noc).
Proteolytic, Cytolytic, Bacteriolytic and various Diastasic Actions of Venoms: Diastasic and Cellular Action on Venoms.
The proteolytic action of venoms on gelatine, fibrin, and egg-albumen has been studied by Flexner and Noguchi,53Delezenne,54and subsequently by Noc55in my laboratory. It was already known thatin vivocertain venoms exert a manifestly dissolving action on the endothelium of blood-vessels and on the muscular tissues themselves.
Delezenne, on his part, has established the existence in snake-venoms of akinaseanalogous to the kinase of leucocytes and enterokinase. Venom alone does not attack egg-albumen coagulated by heat, but it confers an exceedingly strong digestive power on inert pancreatic juices.
Lachesis-venom has been found to be much the richest in kinase. It digests gelatine perfectly, and when this substance has been subjected to its action it is no longer capable of being solidified.
Lannoy,56on the other hand, experimenting upon albuminoidsubstances (casein, albumins of ox-serum) in solution, has shown thatCobra-venom and that ofViperadisintegrate the albuminoid molecule; but the latter remains soluble after the addition of formol and is no longer precipitable by acetic acid. The hydrolysis never leads to the stage of peptone, but only to the formation of albumoses which give biuret-reaction.
The action of venoms upon fibrin may be demonstratedin vitroby bringing sufficient quantities of venom, 1 centigramme, for example, into contact with small fragments of non-heated fibrin, derived from blood clots from an ox, rabbit, or birds, and carefully washed. These fragments soon separate from each other, and become dissolved in a space of time which varies according to the venom used. TheViperine-venoms, especially those ofLachesisandAncistrodon, are the most active.Viper-venom is much less so, and the venoms ofColubridæare the slowest.
This proteolytic action of the various venoms corresponds pretty exactly to their coagulant and decoagulant action on rabbit- or horse-plasma, so that, as I have already stated, we must suppose that the property possessed byViperine-venoms of more or less rapidly dissolving blood which they have caused to coagulate, results from the fact that these venoms contain, in addition to a coagulant substance, another substance which is strongly proteolytic.
The latter is destroyed by heating.Lachesis-venom, when heated to 70° C., no longer has any dissolving action on either gelatine or fibrin. Moreover, antivenomous serum furnished by horses vaccinated against heated venoms does not prevent proteolysis by non-heated venoms. On the other hand, the serum of animals vaccinated againstViperine-venoms, simply filtered by the Chamberland process and non-heated, affords perfect protection to gelatine and fibrin against the dissolving action of these venoms.
Simon Flexner and Noguchi57have observed that the venoms ofNaja,Ancistrodon,Crotalus,Vipera russellii, andLachesis flavoviridis, contain substances which possess the property of dissolving a large number of the cells of warm-blooded and cold-blooded animals, and that thesecytolysinsare very markedly resistant to high temperatures.
They employed for their experiments 5 per cent. emulsions of organs, spermatozoids, or ova in physiological saline solution. The solution of venom at a strength of 1 per cent. was kept in contact with the different kinds of cells for three hours at a temperature of 0° C.; the liquid was then centrifuged and examined with the naked eye and under the microscope.
The venoms experimented upon dissolved more or less rapidly the parenchymatous cells of the liver, kidney and testicle of the dog, guinea-pig, rabbit, rat and sheep. The most active venoms in this respect were those ofVipera russellii,Ancistrodonand theCobra; the venom ofCrotaluswas the least active.
With regard to the nerve-cells, spermatozoids and ova of cold-blooded animals (frogs, fish, arthropods, worms, and echinoderms)Cobra-venom proved to be the most active; then that ofAncistrodon, and lastly that ofCrotalus.
These cytolysins are not destroyed by heating for thirty minutes at 85° C. in a damp medium, nor by dry heating for fifty minutes at 100° C.
If we bring into contact with a 1 per cent. solution ofCobra-venom, rendered aseptic by filtration through porcelain, sensitive micro-organisms, such as the cholera vibrio, or the bacterium of anthrax in a very young non-sporulated culture, or in itsnon-spore-producing variety, we find that these microbes are dissolved by the solution of venom in varying periods of time.
On making a direct microscopical examination we see that Koch’s vibrios become immovable, then break up into granulations and disappear in the liquid. The bacteriolysis is even more distinct in the case of the bacterium. The enveloping membrane seems to dissolve, and the microbe appears as though composed of a series of granulations placed end to end, which finally disperse and disappear.
By my instructions this bacteriolytic property of venom with respect to different species of micro-organisms was studied by Noc. It was especially clearly seen with the non-spore-producing bacterium of anthrax, the cholera vibrio,Staphylococcus aureus, the bacillus of diphtheria, andB. subtilisin a young culture; it is less distinct withB. pestis,B. coli, andB. typhi, is almostnilwith the pyocyanic bacillus andB. prodigiosus, andnilwithB. tuberculosis.
Investigations have likewise been made by Noc, and subsequently by Goebel,58in order to determine whether cobra-venom dissolves Trypanosomes. These hæmatozoa are more resistant than bacteria, but they nevertheless end by being dissolved after twenty to thirty minutes’ contact in the 1 per cent. solution.
The bacteriolytic substance in venom is distinct from that which produces proteolysis, for the latter is destroyed at 80° C., while the former only disappears with a temperature of and beyond 85° C. maintained for half an hour. It is likewise distinct from the hæmolysin, for this resists temperatures considerably higher than 85° C. Moreover, venom which has dissolved microbes until the saturation point has been reached, is found to have preserved in its entirety its hæmolytic power upon the red corpuscles of the horse. Neither does it act upon the microbes owing to the presence of acytaseoralexin. The well-known characteristics of alexins are not met with here—destruction at 55° to 56° C., sensitivity to light, rapid alteration at ordinary temperatures, &c.
We cannot, again, compare the bacteriolytic action of venom to that of rat-serum, which dissolvesB. anthracisby aid of a substance distinct from vibrionicide alexin. According to the researches of Malvoz and Y. Pirenne, the lysin of rat-serum appears to be a basic substance, the neutralisation of which destroys its activity. NowCobra-venom in a very active solution is perfectly neutral to sensitive litmus papers, while these are turned blue by rat-serum. Moreover, venom acts not only upon microbes of the same kind, but also on very different species which are not affected by rat-serum, especially uponB. pestis, for which, on the contrary, this serum, when fresh, proves a favourable culture medium. The bacteriolytic power ofCobra-venom therefore constitutes a special property of venom.
“In their work on the cytolysins of venom, S. Flexner and Noguchi have shown that animal cells, when heated to 55° C. and rendered inactive, do not undergo complete dissolution under the influence of venoms which destroy the fresh cells. The authors in question infer the existence of cellular receptors (endo-complements, according to the theory of Ehrlich), which fix the amboceptors of venom. Pursuing the same order of ideas, I have observed that bacteria killed by heating for one hour at 60° C. do not undergo total disintegration as do living bacteria. But, while Flexner and Noguchi infer the plurality of the cytolysins in venom for different animal cells, I have not been able to prove the same thing with regard to the bacteriolysin; venom saturated with cholera vibrios to such an extent that vibrios added at repeated intervals are no longer dissolved, is incapable of dissolving another highly sensitive species of microbe, such as the asporogenous bacterium, andvice versâ. Besides, it would be difficult to understand the existence in venom of cytolysins specific for a whole series of species of micro-organisms” (Noc).59
Antivenomous serum, in a dose of 0·01 or 0·05 c.c., neutralizes the bacteriolytic action of 1 milligramme ofCobra-venom,while normal serum heated, even in larger doses, is without effect. The lysin and the antivenomous serum appear also to enter into stable combination; by heating to 80° C., after dilution of the mixture neutral antivenomous serum + venom, the property of dissolving is not restored to the latter.
Pursuing his researches upon the bacteriolytic actions, Noc has also shown that thefreshserums of the rabbit, horse, guinea-pig, rat, and man are capable of destroying them completely. We must conclude from this that venom has the property of fixing the alexin of these fresh serums, and in fact it is easy to show that this fixation takes place by experimenting with hæmolytic alexin, which is much more easy to study; it is sufficient to eliminate the intervention of the hæmolysin proper toCobra-venom.
With this object, Noc employed horse-corpuscles (which are readily dissolved by fresh rat-serum), and neutralised the hæmolysin proper to the venom by antivenomous serum, which has no effect upon fresh horse-corpuscles and upon the alexin of rat-serum.
For experimental purposes six tubes are prepared with contents as follows:—
(1) 0·5 c.c. of fresh rat-serum.
(2) 0·5 c.c. of fresh rat-serum + 0·5 milligramme of Cobra-venom (0·5 c.c. of a solution of 1 in 1,000).
(3) 0·5 c.c. of fresh rat-serum + 1 milligramme of venom (after fifteen minutes’ contact of the venom with the alexin in tubes 2 and 3 the venom is neutralised by 1 c.c. of antivenomous serum in the case of tube 2, and by 2 c.c. in that of tube 3).
(4) 1 milligramme of venom.
(5) 1 c.c. of antivenomous serum.
(6) 0·5 c.c. of fresh rat-serum + 1 c.c. of antivenomous serum.
To each tube 2 drops of defibrinated horse-blood are added, and the tubes are placed in the stove at a temperature of 35° C.
In tubes 1 and 6, which contain fresh rat-serum alone, and fresh serum + antivenomous serum, hæmolysis appears in a few minutes.In tube 4, which received venom alone, hæmolysis is also produced in one hour. It is not produced at all in tubes 2 and 3, which received the neutral mixture of fresh serum and venom, proving that the hæmolytic alexin has been fixed by the venom. The latter, therefore, here plays the part of a true fixator oramboceptor.
Venom behaves, in short, after the manner of extracts of organs. The fixation of hæmolytic alexin by extracts of organs, the tissues, and animal cells (liver, spleen, spermatozoids, &c., &c.), has already been demonstrated by V. Dungern, P. Müller, Levaditi, and E. Hoke. The same fact is also observed with solutions of peptone. The fixation of alexin is therefore a general property of certain albuminoid molecules.
It was interesting to endeavour to reproduce, withCobra-venom, J. Bordet’s experiments upon alexins and anti-alexins. It was to be hoped that we had in this substance an anti-alexic body capable of being preserved for an indefinite time and constant in its activity, which would enable us easily to measure the dose of alexin contained in a small quantity of a serum, or other liquid of leucocytic origin.
The experiment proved to Noc that, contrary to the ideas of Ehrlich and his pupils, and conformably to the results obtained by Bordet with serums and toxins, the neutralisation of venom takes place in a variable ratio.
If a dose A of fresh serum is capable of neutralising exactly 5 milligrammes ofCobra-venom with regard to a sensitive microbe, on employing a dose of the strength of 2 A we ought to find a bactericidal dose, 1 A, in the excess of serum, according to the theory of definite proportions. No such bactericidal action is seen, however; the serum, on the other hand, acts in the contrary direction by means of its nutritive substances, and in the mixture 2 A +venomwe obtain a larger number of colonies of micro-organisms than in the mixture A + venom.
We see, then, that the property of cells of fixing in excess the active substance in serums, discovered by Bordet for the hæmolysins(staining phenomena), is met with again in the case of extracts of organs, at least with regard to the bacteriolytic substance ofCobra-venom.
It results, then, from the foregoing facts thatCobra-venom contains a cytolysin, which acts upon micro-organisms and is capable of fixing the alexin of normal serums.
The application of these data to the living animal is evidently full of difficulties, by reason of the complexity of the substances that come into play. Let us see, however, to what extent they are capable of serving to explain the phenomena that are produced as the result of poisoning.
It was observed by Kaufmann that the cadavers of animals which have died from snake-bite are very rapidly invaded by the bacteria of putrefaction. Welch and Ewing, referring to these phenomena of rapid putrefaction in cases of death from venom, explained them as being due to the loss of the bactericidal power of the serum. In hot countries, even when snake-bites are not fatal, they are frequently complicated by local suppuration or gangrene, occasioned by micro-organisms introduced at the time of the bite. The minute analysis of the phenomena of poisoning shows, in reality, that the organism undergoes different modifications according to the quantity of venom injected and its channel of penetration.
When the dose of venom is rapidly lethal, whether because it penetrates into the veins or because a larger amount of it is diffused beneath the skin, it occasions a transient hypoleucocytosis, which is, moreover, a reaction common to injections of venom, pro-peptone, extracts of organs, and microbic toxins (Delezenne, Nolf). It follows that blood collected a short time after the injection may be totally bereft of its bactericidal power, in consequence of the disappearance of the leucocytes, which have migrated into the organs.
Thus it was observed by S. Flexner and H. Noguchi that the serum of a rabbit, treated with 10 milligrammes ofCobra-venom, showed, fifty-seven minutes after the injection, a great loss of bactericidal properties. But it is impossible to conclude, fromthe diminution of bactericidal power in this experiment, that the alexin becomes fixed by the venom. Since the secretion of alexin is connected with the presence of leucocytes, the hypoleucocytosis due to the venom is sufficient to explain the loss of bactericidal power.
Nevertheless, the action of venom is not confined to these physiological phenomena; in diffusing itself through the organism it stays more especially in parts where the circulation has become slower, in the capillaries of the organs where the leucocytes that have disappeared from the general circulation are already to be found agglomerated and altered. Here the cytolysins of the venom, continuing their effects, are capable of neutralising the alexins set at liberty by the destruction of the leucocytes, and thus the rapid multiplication of the bacteria of putrefaction, which have come from the intestine or were carried in with the bite, is easily explained. In the same way, we can account for the suppuration that is met with as a complication of non-lethal bites, in spite of the hyperleucocytosis consequent upon the penetration of a weak dose of venom; immediate neutralisation of the alexin set at liberty at the level of the wound has sufficed to enable micro-organisms to multiply.