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.

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.

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.