Bacteria in Daily Life

A correct calculation of the quantity of venom required in every case was, however, found to be quite impossible, for so virulent is the poison that a single drop of an emulsion produced by pounding up eight glands in 300 grammes of distilled water is sufficient, when introduced into the vein of a rabbit's ear, to kill it in five minutes. All the mammals to which Calmette administered this cobra venom, such as monkeys, dogs, rabbits, guinea-pigs, rats, succumbed more or less quickly, according to the size of the dose.

Small birds and pigeons die very rapidly, but the domestic fowl is more fortunate, being somewhat less susceptible. Frogs also fall a prey to the venom, but they are far more refractory than rabbits, for it takes thirty hours to kill a frog with a dose of venom which would infallibly destroy a rabbit in ten minutes. Toads, curiously, do not enjoy to the same extent this power of resisting its toxic action, for they die more quickly than frogs, whilst it makes short work of lizards and chameleons. Fish form no exception to the rule, and even invertebrates, such as leeches, are killed by minute traces of venom.

Whilst Calmette has found that the venom of different kinds of reptiles exhibits marked differences in its toxic character, he has also discovered that the venom secreted by one and the same serpent varies considerably, according to the length of time the animal has fasted. He describes how he kept a naja haje (Cleopatra's asp) in his laboratory, which during the whole eight months that it lived never took any food whatever, although it was offered the most diverse dainties. On its arrival it was made to bite on a watch-glass, this being one method adopted for collecting the venom; the liquid was at once dried, and 0·7 milligramme was found to kill a rabbit weighing nearly four pounds in four hours. Two months later on, when the venom was again collected, 0·25 milligramme proved a fatal dose. On the death of the animal, at the end of eight months, the venom extracted from the glands was so toxic that it only required 0·1 milligramme to kill a rabbit of about the same weight as the previous one. The same curious fact was noted in the case of a cobra's venom. Another circumstance which appears to control the degree of toxicity inherent in serpent venom is the interval of time which elapses between two successive bites. The longer the interval the more virulent is the venom; and Calmette points out that these observations are in accordance with what has for a long time been known in France with respect to indigenous vipers – that their bites are far more dangerous and far more fatal in the spring, after the winter period of torpor is over, than in the autumn.

Until quite recently it was thought that the only creatures which could resist the fatal action of this poison were serpents, both poisonous and non-poisonous. Calmette was led to this conclusion because, although he inoculated large doses, as much as ten drops, into cobras, they suffered absolutely no inconvenience, and the same results were obtained with harmless snakes. On repeating these experiments, however, and using much larger quantities of venom, Calmette has found that they do ultimately succumb. That their susceptibility in comparison with other animals is very slight, may be gathered from the fact that a lethal dose of venom for reptiles is roughly estimated to amount to as much as three times the quantity of venom normally present in their respective poison glands. These animals, therefore, although very refractory, are not absolutely immune from the action of venom-toxin.

There are, however, other animals which enjoy a relative although not absolute immunity to snake poison, and amongst these may be mentioned swine, hedgehogs, and the mongoose. Swine, it is well known, will greedily devour reptiles, and in some countries they are specially trained up and employed for this purpose. Of particular interest, however, are some experiments which were carried out to test the traditional immunity towards this toxin ascribed to the mongoose. These animals are very useful in sugar plantations, and are largely employed to keep down the serpents and rats with which they abound, for the carnivorous little mongoose is extremely partial to such prey. Attempts have been made by sugar planters to introduce them into Martinique, where they are not found in the wild state, as in the island of Guadeloupe.

Six specimens of the mongoose were forwarded to Calmette from Martinique, and these particular animals, it was stated, had never been set at liberty since they were imported, so that they had had no previous experience of snakes or venom. On arriving at the laboratory, one of these little creatures was placed in a glass cage along with a large cobra. The cobra, at once rising up and dilating its neck, darted with fury upon the mongoose; but the latter, thanks to its extraordinary agility, escaped being caught, and took refuge, stupefied and terrified for the moment, in a corner of the cage. This stunned condition, however, did not last long, for just as the incensed cobra was preparing to make a fresh attack upon its insignificant little victim, the latter, with wide-open mouth, rushed and jumped upon the head of its enemy, viciously bit through its upper jaw, and broke its skull in a few seconds. Thus, although in size but a little larger than a squirrel, this tiny creature was more than a match for a cobra two yards long.

Artificial inoculations of cobra venom into the mongoose fully substantiated all the observed facts as to its remarkable immunity from this poison. A dose sufficient to kill a large rabbit in three hours was absolutely without effect; only when the venom was introduced in quantities amounting to as much as eight milligrammes was it followed by fatal results. Thanks, therefore, to their extraordinary agility and remarkable power of resisting the effects of this lethal toxin, these little animals are able to battle successfully with the most dangerous reptiles.

The rapidity with which serpent venom becomes absorbed by the system is almost incredible, and is well illustrated by the following experiment. A rat was inoculated with venom near the tip of its tail. One minute later the latter was cut off a short distance above the point of inoculation; but this operation was quite unable to save the animal's life, for even in that brief interval the poison had accomplished its fatal work, and a few hours later claimed its victim.

This rapid diffusion of the venom helps to explain the difficulty which is experienced in arresting the course of the poison by local treatment, for its passage is too rapid to permit of its being overtaken by superficial measures of even the most stringent character. But Calmette points out that local precautions are not to be neglected, for although they cannot nullify the action of the venom, they undoubtedly do delay its progress, and thus create a longer interval or respite, during which an opportunity is afforded for administering the anti-toxin. Before, however, passing on to the investigations which have culminated in the production of a specific antidote for this terrible toxin, there are a few more details which Calmette has furnished as to its character which are of interest. Serpent venom is characterised not only by its intensely virulent properties, but also by the tenacity with which it retains them under diverse circumstances. Thus it may be stored up for a whole year, and yet at the end of that time be as active as ever; and even after several years, although its toxic powers are somewhat reduced, it still retains them to a very appreciable extent.

Unlike the bacterial toxins, this venom toxin can stand exposure to considerable temperatures without injury to its activity, and that of the cobra only suffers after it has been submitted to 98 °Centigrade for twenty minutes. Sensitiveness to temperature varies, however, with the snake from which the venom is derived. Thus the venom of the so-called "tiger-snake" of Australia will stand being exposed for ten minutes to from 100° to 102° degrees Centigrade, and its virulence only disappears when this temperature has been applied for twenty minutes. The venom of the "black snake," another Australian variety, loses its toxicity at a temperature of between 99° and 100 °Centigrade; whilst an exposure to only 80 °Centigrade for ten minutes is sufficient in the case of viper venom, according to Messrs. Phisalix and Bertrand, to profoundly modify its lethal action. A continuous exposure for a fortnight to a temperature of 38 °Centigrade does not affect cobra venom in the least; but if during that same time it has been placed in the sunshine, it entirely loses all its lethal properties. Thus, a pigeon was inoculated with about thirty drops of venom which had been exposed to the sun's rays for fourteen days, and it survived; whilst another pigeon was inoculated with a little over six drops of similar venom which had been kept during this time in the dark, and it died in a quarter of an hour.

All these elaborate researches as to the character of serpent venom were essential to enable the next step to be taken in the elaboration of the antidote. Before this great achievement could be accomplished it was necessary to first succeed in artificially immunising animals against the effects of this powerful toxin, so that the serum of such animals could be applied for the protection and cure of other animals from the effects of snakebites.

It may be readily conceived that the task of artificially rendering animals immune from snake poison was not an easy one, for the process depends upon training the animal to gradually withstand larger and larger doses of the venom; and considering the intensely toxic character of the substance which had to be handled, the danger was ever present of the animal succumbing to venom poison before its serum had acquired the requisite pitch of protective power to render it of service as an anti-toxin. Dr. Calmette tells us that he carried out a very large number of experiments before he met with success. But it is not necessary here to discuss his various efforts; suffice it to say that at length his labours were rewarded, and the following extract from one of his memoirs describes the methods which he adopted for this purpose: —

"The best method of procedure for the purpose of vaccinating large animals destined to produce anti-venomous serum consists in injecting them from the outset with gradually increasing quantities of the venom of the cobra mixed with diminishing quantities of a one-in-sixty solution of hypochlorite of lime.8 The condition and the variations in the weights of the animals are carefully followed, in order that the injections may be made less frequently if the animals do not thrive well. Quantities of stronger and stronger venom are in turn injected, first considerably diluted, and then more concentrated; and when the animals have already acquired a sufficiently perfect immunity, the venoms derived from as large a number of different species of snakes as possible are injected. The duration of the treatment is of considerable length – at least fifteen months – before the serum is sufficiently active to be used for the purposes of treatment."

An immense number of animals have been vaccinated by this method at the Pasteur Institute at Lille, where Dr. Calmette is now director; and in one of his memoirs we are told that they have horses there which have yielded during a period of eighteen months serum extremely active against venom. These horses receive in a single inoculation, without suffering the least inconvenience, doses of venom sufficient to kill fifty horses fresh to the treatment.

Large quantities of this serum have been forwarded from the Lille Institute to various parts of the world where venomous serpents are most frequently met with, and already important evidence has been collected as to its efficacy in cases of human beings bitten by dangerous reptiles. So impressed with its importance are Indian medical authorities, that its preparation has been included in the work which the new great bacteriological institute at Agra is carrying on.

The importance of the production in situ of this anti-venomous serum has been recently demonstrated by the experiments which have been conducted in the Plague Research Laboratory, Bombay, by Mr. Lamb and his colleagues, on the keeping properties of such serums in India. From the careful investigations which have been made on this subject, these gentlemen state that anti-venomous serum undergoes a progressive and fairly rapid deterioration when stored in hot climates, and that this deterioration is greater and more rapid the higher the mean temperature to which it is subjected.

The protective potency of this horse-serum may be gathered from the fact that it suffices to inject a rabbit, for example, with a quantity amounting to about one two-hundred-thousandth of its weight to ensure the latter acquiring complete immunity from a dose of venom capable of otherwise killing it in twelve hours.

The rapidity with which it acts is also extremely remarkable. Thus, if a rabbit receive two cubic centimetres (about fifty drops) of anti-venomous serum in the marginal vein of one of its ears, it will suffer with absolute impunity an injection of venom into the marginal vein of the other ear capable of killing it under ordinary circumstances in a quarter of an hour. Its curative powers are not less remarkable, for it is possible to inject venom sufficient to kill an animal in two hours, and to let one hour and three-quarters elapse before administering the antidote, and yet at this late stage to save the victim's life, although it is necessary where such a long interval has occurred between the respective venom and serum injections to employ the latter in larger quantities than is usually required. Dr. Calmette believes that the anti-toxin may be applied at an even more advanced stage of the disease if it is employed in yet larger doses. Another novel and important feature about this anti-venomous serum is the fact that it not only protects animals from one species of very active venom, such as that of the cobra and other poisonous snakes, but it also affords protection from the dreaded venom of scorpions. This is a very remarkable and significant discovery, for hitherto the opinion has been stubbornly held that each toxin requires its specific anti-toxin for its correction. Dr. Calmette has, however, frequently indicated by his researches that this view cannot be considered so completely proven as is claimed by its supporters, and his latest investigations support the theory that particular toxins may be counteracted by several anti-toxins of different origin. Thus it has been shown by Calmette and Roux that rabbits hyper-vaccinated against rabies acquire the power of resisting venom-poison, and that the serum of horses vaccinated against tetanus or lock-jaw also nullifies the action of serpent venom.

The practical bearing of this discovery is obvious, and the hope is justified that the at present cumbrous appliances required for the elaboration of anti-toxins of such varied origin will ultimately give way to simpler and less costly methods, which will admit of these new antidotes being more widely circulated and applied.

We have seen that although most animals fall an easy prey to serpent venom, yet there are a few notable exceptions, amongst which may be mentioned hedgehogs, swine, and the mongoose. Now the very natural question arises why, if these animals are already in such a high degree immune from this poison, should not they be employed to furnish forth protective serum, instead of laboriously training up susceptible animals to become artificially immune and supply this venom anti-toxin?

This brings us face to face with one of the many problems connected with the subject of immunity which so far have successfully eluded all attempts made to solve them. Experience has shown repeatedly that although artificially acquired immunity from a particular poison can be handed on by means of an animal's serum, yet the natural immunity from a given poison enjoyed by one species of animal cannot be similarly transferred to less-favoured varieties.

This fact has long been recognised in the case of poisons of bacterial origin. Thus, white rats are absolutely immune from diphtheria, but Wassermann showed some years ago that the serum of these animals has no power whatever to counteract the action of diphtheria-toxin in other animals. Guinea-pigs were inoculated with fatal doses of diphtheria toxin along with white-rat serum; but although other guinea-pigs treated with the same toxin mixed with the ordinary artificially elaborated anti-diphtheritic serum survived, those which received the rat serum died in every case.

Now very similar results have been obtained by Calmette in respect to the serum of animals naturally immune from serpent venom. The serum of the refractory little mongoose, as well as that of the hedgehog, is wholly unable to save other animals from the lethal effect of venom poison, and similar results have been noted in respect to swine serum. But a very curious fact has also been discovered by Calmette —i. e. that these so-called naturally immune animals very frequently are quite incapable of being artificially trained to elaborate a serum possessing protective powers which can be transferred to another animal.

How splendid a domain for beneficent research lies before the scientific investigator is apparent to all, and the important work already accomplished is but an augury of yet greater discoveries awaiting the labours of such leaders as Calmette. It is not surprising, therefore, that the scientific interest in toxins and anti-toxins shows no signs of abatement. On the contrary, the competition for obtaining and working the new "claims" which pioneer research enthusiasts are constantly engaged in "pegging out" remains as keen as ever.

Despite, however, the extraordinary interest which this subject has aroused in scientific circles all over the world, nearly ten years elapsed before any notice was taken of the curious discovery made by two brothers that the blood of eels contained a highly poisonous principle, and the memoir containing this remarkable announcement remained until comparatively recently buried in the Italian journals where it was first published.

Calmette was, we believe, the first to call attention to this discovery of the brothers Mosso and give it the prominence it deserves, and both he and other investigators have not only fully confirmed it, but have greatly added to our knowledge concerning the character of the poison contained in eel serum.

Now the venerable Izaak Walton, in one of his quaint and most fascinating discourses, which although written more than two centuries ago have a freshness as if penned but yesterday, waxes enthusiastic over the eel, and supplies an elaborate recipe for its preparation for the table, telling us "it is agreed by most men that the eel is a most dainty fish; the Romans have esteemed her the Helena of their feasts, and some the queen of palate-pleasure." The announcement that the blood of eels is poisonous will hardly, despite its scientific interest, form a comfortable subject for reflection to the modern votaries of this novel Helena. Indeed, in the present timid temper of the public, this article of diet would not improbably share the ill-odour which befell the unfortunate oyster and be practically banished from our tables; but although the oyster is perhaps justifiably at present ostracised from our menus, taking the majority of its breeding-grounds into consideration, it would be the height of injustice to measure out similar drastic treatment to the eel.

That the oyster bred in sewage-contaminated beds may revenge itself upon its consumer by infecting him with the germs of typhoid has been repeatedly contended, but that the eel, although its unsavoury surroundings are proverbial, can be held responsible for poisoning those who eat it has never, we believe, been seriously maintained, although there is an old Italian saying which bids us "give eels and no wine to our enemies."

Public confidence, however, in the eel as an article of food need not be shaken, for it is satisfactory to learn that researches which, on the one hand, condemn eels as living generators of a highly poisonous substance, on the other hand allay any alarm which they may have reasonably raised by showing that this toxic principle is entirely destroyed in the processes of digestion, and that, therefore, taken through the mouth it is rendered harmless, and only when introduced into the system by inoculation beneath the skin or injected into the peritoneum can it assert its dangerous properties. That the blood of eels is, however, justifiably to be in future classed amongst the toxins, the number of which has of late been so increased, is at once apparent when we learn that about a dozen drops inoculated into a dog weighing about fourteen pounds will destroy the latter in less than ten minutes, whilst pigeons, rabbits, and guinea-pigs similarly treated, only with smaller quantities, also invariably succumb to its lethal action.

Quite recently an endeavour has been made to determine precisely the degree of toxicity possessed by eel's blood, or, in other words, to standardise the poisonous principle contained in it, so as to afford a guide to those experimenting on the subject; and it has been asserted that one cubic centimetre, or about twenty drops, injected into the veins of a rabbit weighing four pounds, may be regarded as a fatal dose for such an animal. But many difficulties surround such an attempt to exactly define the degree of toxic action possessed by such a substance, for, in the first place, the blood varies in respect to this property in different eels, whilst it also differs widely in character at different stages of the life of the fish. This seasonable variation in toxic character has been noticed in the case of viper venom, which it will be remembered was shown to be far more lethal in action when collected from snakes in the spring of the year than in the winter months.

The toxic substance contained in eel serum was originally called by its discoverers, the Mosso brothers, ittio-tossina; and they record the fact that the blood of rabbits and frogs, which animals had succumbed to its action, did not coagulate after death, whilst, curiously, in the case of dogs this abnormal phenomenon was not observed.

There are various means which may be resorted to for destroying the poisonous principle contained in eel blood, and from a dietetic point of view it is satisfactory to know that heat-exposure for a quarter of an hour to a temperature of from 57·7° to 77·7 °Cent. entirely removes it, whilst its virulence is greatly modified by submitting it for a longer period, twenty-four hours, to a much lower temperature, i. e. 37 °Cent. It also gradually loses its toxic properties eight days after it has been collected, even when carefully shielded from light, a feature which contrasts favourably with viper venom, which can be kept for more than a year and remains as active as when first derived from the snake. We have seen also that its toxic properties invariably succumb to the processes of digestion, so that even if fashion or fad or advertising speculators, backed by scientific names, were to decree that a wealth of nourishment and support was contained in raw eel "juice," and the edict went out that it was a desirable and highly important article of invalid diet, the general public may, according to its wont, innocently accept the edict and in this case suffer no evil consequences.

But another and very remarkable method of mitigating the virulence of eel blood, and one which so far has received no explanation, is mentioned by Dr. Wehrmann, of Moscow, who has been lately studying the character of this fish's blood in Dr. Calmette's laboratory at the Pasteur Institute at Lille. Dr. Wehrmann found that if blood serum be taken from animals previously rendered artificially immune to the action of serpent venom, and if some of this so-called anti-venomous serum be injected under the skin of eels some hours before they are killed, the lethal properties of their blood after death are considerably reduced. Thus, an eel weighing about six ounces received subcutaneous injections of five cubic centimetres of anti-venomous serum; after the lapse of four-and-twenty hours it was killed and bled, and its serum inoculated into animals in the usual way. But whereas two cubic centimetres of normal eel blood sufficed to kill a guinea-pig, this eel's blood had to be administered in twice that quantity to produce a fatal result, so that its toxic character had been reduced to a very appreciable extent. The readiness with which eel serum parts with its lethal properties, and the restricted conditions under which they can operate, sufficiently assure us that in the present state of our knowledge there is no danger to be apprehended from this fish, and in the absence of any experiments to show what is the effect on human beings of subcutaneous inoculations of such blood, there is no call for this substance to be scheduled under the Poisons Act. We have, however, by no means exhausted the extremely curious properties which characterise this material, and these properties are brought to light in a remarkable manner in connection with the investigations which have been carried out to artificially protect animals from its lethal influence, and also in some interesting experiments which have been made to compare the toxicity of eel blood with that of vipers.