Although scientific men succeeded only a little more than ten years ago in vaccinating against poisons by artificial methods, savage races and ancient peoples at a very remote period undoubtedly possessed methods of counteracting the effects of certain venomous substances. The frequent observation of cases in which doses of poisons, insufficient to cause death, brought about a more or less durable resistant condition, must result in the elaboration of artificial means of preventing the intoxications.

Von Behring^[521] points out that analogous facts must have been known to the physicians of ancient times; and it is in such knowledge that we must look for the source of the dogma put forward by Hippocrates, that the factor which produces a disease is also capable of curing it.

To Pliny we are indebted for the now well-known story, that Mithridates of Pontus possessed the means of protecting himself against various poisons by a process of adaptation, and, amongst others, by the use of the blood of Pontine ducks to which he had given poisons by the mouth.

The adaptation of horses and of the highlanders of Styria to arsenic, as well as that of the many morphinomaniacs to morphia, is known to everybody. A man, habituated to morphia, is able to consume daily a dose several times the fatal one; indeed, cases have been known of people acquiring the power of consuming two, and even three, grammes of morphia per diem. Man may acquire an adaptation to toxic substances of the most diverse character, such as arsenic, alcohol, morphia, nicotine, etc.

Even when we had obtained much information concerning acquired immunity against micro-organisms we still knew nothing of the mechanism of such adaptation, or as to the possibility of acquiring a special immunity against bacterial poisons. Charrin and Gamaleia’s discovery that animals vaccinated against a micro-organism are just as susceptible to its toxic products as normal animals, led Bouchard^[522], in whose laboratory it was made, to say that the idea of the adaptation of cells to bacterial poisons must be dropped. He developed this thesis at the International Congress at Berlin in 1890, and formulated it as follows: “When we inject a healthy animal and a vaccinated one with the soluble products of the micro-organism which has been used for the vaccination, the dose required to kill each animal is exactly the same. Let us not speak, then, of the training of the leucocytes, and of the adaptation of the nerve cells to bacterial poisons: it is pure rhetoric.” At this time we had only just commenced to acquire exact knowledge concerning the toxins of micro-organisms. For a considerable period they were sought for amongst the ptomains, very stable substances allied to the alkaloids; here, however, we were working in a wrong direction. It was not until the classic researches of Roux and Yersin^[523] on diphtheria toxin, published in 1888 and 1889, that the true nature of bacterial poisons was revealed. It was found that we were not dealing with ptomains, but with soluble ferments, substances of indeterminate chemical composition, allied to the albuminoids, and, like them, unstable. The methods adopted by Roux and Yersin in their study of diphtheria toxin enabled other investigators to discover the analogous toxins of several other bacteria. Knud Faber^[524] and Brieger and Fränkel^[525] soon succeeded in separating the toxin from the tetanus bacillus, a toxin capable of producing in animals tetanic contractions as typical as those obtained with cultures of the tetanus bacillus.

These investigations inaugurated a new era in microbiology and enabled us to attack the problem of acquired immunity against bacterial toxins scientifically. Within a few months of the declaration made by Bouchard at the Berlin Congress, there appeared, almost simultaneously, the earliest publications on the possibility of vaccinating laboratory animals against the toxins of diphtheria and tetanus by artificial methods. Immediately after the discovery of these poisons, the attempt was made to immunise various species of animals against them, but here very great difficulties were met with; the animals, after receiving increasing doses of toxin, became thin and ultimately died. It occurred to Fränkel^[526] that the toxic action of the diphtheria poison might be weakened by subjecting it to a temperature of 60° C. Independently, von Behring and Kitasato^[527] used chemical substances, especially iodine trichloride, to attenuate the action of the tetanus and diphtheria toxins. The animals which resisted these modified poisons were found to be capable of tolerating gradually increasing doses of unaltered and very active toxins. By the use of these methods it was found possible to obtain a definite and lasting immunity against these microbial products.

The discovery of the possibility of vaccinating against bacterial toxins was soon followed by the demonstration of the antitoxic power of the blood of animals that had acquired such artificial immunity against these poisons. Everyone knows of and appreciates von Behring and Kitasato’s great discovery. It opened up a new and fruitful field of research from most diverse points of view. Ehrlich^[528] was able to apply it to the vaccination of animals against the vegetable poisons ricin, abrin and robin, and thus to establish rigorous methods of immunisation and to obtain very important results concerning immunity against toxins in general. He also succeeded in demonstrating that animals vaccinated against these vegetable poisons, which, by their nature, approximate to the microbial toxins, develop in their blood a most marked antitoxic property.

Some years later, the discovery of antitoxins was extended to snake venoms, poisons of animal origin which, like the vegetable poisons studied by Ehrlich, present a chemical composition analogous to that of the microbial toxins. Phisalix and Bertrand^[529] and Calmette^[530], working independently, discovered methods of vaccination against snake venom and were able to demonstrate the existence of an antitoxic power of the blood in immunised animals.

The works above briefly referred to gave us the fundamental basis of our present knowledge on acquired immunity against toxins.

It would be very interesting to be able to determine whether the lower animals can be vaccinated against the toxic substances to which they are susceptible. Unfortunately in the study of this problem we encounter very great difficulties. Making use of various methods I have often tried to solve it. The crayfish is susceptible to snake venom and to the ichthyotoxin of eel’s serum, and I have tried at various times to vaccinate it against these poisons. The results, however, were so inconstant and even contradictory that I was unable to draw any definite conclusion from them.

It is, indeed, very difficult to vaccinate the lower vertebrata against poisons. Several attempts have been made in my laboratory to immunise frogs against tetanus toxin, but without success. Calmette and Deléarde^[531] obtained the best results with abrin. They succeeded in vaccinating frogs—which are not very susceptible to this vegetable toxin, though they are far from presenting a real natural immunity—against doses which are absolutely fatal for the control animals. These observers, however, had to proceed very cautiously, and they allowed a very long interval between each injection of abrin. The blood of their vaccinated frogs not only did not prove to be antitoxic against abrin, when injected into mice, but for long retained sufficient of this toxin to poison normal mice. This experiment certainly tells against the hypothesis that the acquired immunity of frogs is due to the development of a specific antitoxic power in their body fluids, but it does not settle the question definitely since it may be objected that the blood, whilst toxic for mice, might, still, be antitoxic for the frog. The antitoxin of this blood might merely be incapable of neutralising all the abrin present. Fresh investigations, then, are necessary.

Even in the higher vertebrata, it is often very difficult to obtain a real vaccination against the various toxins. In the small mammals, which exhibit a great susceptibility to these poisons, it is specially difficult to obtain an artificial immunity. As Vaillard and von Behring have demonstrated, it is possible to vaccinate such animals by means of gradually increasing doses of unmodified toxins, but this method demands much time, is often dangerous, and hence is not very practical. Poisons that act through the alimentary canal are the most serviceable for vaccination, as has been demonstrated by Ehrlich. This investigator had to abandon the vaccination of mice by means of subcutaneous injections of ricin on account of the sloughing set up at the point of inoculation. He then had recourse to vaccination by way of the mouth, which gave very good results, not only with ricin but also with abrin. This mode of vaccination, however, is applicable to a small number of poisons only.

We can also vaccinate mammals, even laboratory rodents, such as rabbits and guinea-pigs, by means of unmodified snake venom, but this method is a very delicate one and must be carefully watched. It is necessary to begin with very small doses of venom, continue them for some time, and increase the amount of venom injected very slowly. Calmette^[532] modified this method by inserting, below the skin and leaving it there, a piece of chalk impregnated with small quantities of venom and surrounded by collodion through which the venom diffuses very slowly and continuously.

Large mammals, sheep, oxen and horses, can be more easily vaccinated by means of unmodified toxins, but they also require to be treated with very special precaution. Salomonsen and Madsen^[533] have given the history of their horse, immunised with diphtheria toxin. Into a mare weighing 665 kilos they were able to inject at the commencement only 1 c.c. of this toxin, and the dose had to be increased very carefully.

In the presence of all these difficulties in the use of unmodified toxins for vaccination, a different method is now generally adopted in the immunisation of animals, small or large, for the purpose of scientific research or for the preparation of toxins on a commercial scale. Vaccination is commenced with toxins modified by heat or by chemical substances. The diphtheria and tetanus toxins, those most employed in the serotherapeutic industry, are subjected to various degrees of heat. Fränkel^[534] was the first to make use of this method for vaccination against diphtheria, and Vaillard^[535] for vaccination against tetanus. It consists in introducing large doses of filtered cultures, heated to progressively lower degrees of temperature, 60°, 55°, 50° C., and then giving gradually increasing quantities of filtered cultures whose toxicity is unaltered. This method is very convenient for small animals, but for large mammals it is greatly simplified by injecting for a certain period toxins heated to 60° C., and, later, replacing these by unmodified toxin.

Phisalix and Bertrand^[536] applied an analogous method to the vaccination of the guinea-pig against the venom of the viper. This poison, which resists much higher temperatures than do the tetanus and diphtheria toxins, received a preliminary heating to 80° C. in order that it might be inoculated without danger into small animals. Under these conditions it confers a certain immunity, but even when heated to 80° C. it, in many cases, still remains sufficiently active to produce fatal results. For this reason, in the vaccination of animals for the preparation of antivenomous serum on a large scale, Calmette had recourse to another method, that of attenuating the venom by means of chemical substances.

Von Behring and Kitasato^[537] were the first to make use of iodine trichloride in the vaccination of animals against the toxins of tetanus and diphtheria. In their early experiments this substance was injected before the toxins were introduced. Later, the mixture was made in vitro and then injected into the animals. Roux devised another method which had the advantage of being simple, certain, and easily employed, for which reason it was soon introduced into commercial and scientific practice. It consists in the injection of mixtures of the tetanus or diphtheria toxins with Lugol’s iodo-ioduretted solution. The iodine, in small doses, instantly neutralises or modifies these poisons and is itself borne well, even by small animals. By employing progressively increasing doses of these mixtures, in which the amount of iodised solution becomes smaller and smaller compared with that of the toxin, we are able, without difficulty, to vaccinate the most susceptible animals and enable them to withstand considerable doses of the pure toxin. By this method it is possible to immunise guinea-pigs against the most active tetanus toxin. The method serves equally well for the preparation of horses for injections of unmodified toxins. For a longer or shorter time (according to the susceptibility of the horse) toxins which are mixed with Lugol’s iodised water are injected. Having made sure of the resistance of the horse, larger and larger quantities of pure, unmodified toxin may be introduced with impunity.

For the immunisation of mammals of all sizes (guinea-pigs, rabbits, dogs, horses) against snake venom, Calmette, in his work at Lille, also makes use of venom modified by chemical substances, but his method differs from those we have just described. During several weeks he injects increasing quantities of venom, mixed with decreasing quantities of a solution of 1:60 of hypochlorite of lime. After this treatment the animals become capable of tolerating fatal doses of unmodified venom and can be injected with larger and larger doses.

In recent years a method of vaccinating horses against certain microbial toxins, and especially against the diphtheria toxin, by means of mixtures of toxin and antitoxic serum, or with these two products successively, has been introduced. Babes^[538] was the first to extol this method as the best for obtaining a high and durable immunisation. Afterwards, several other observers, amongst whom I may cite Pawlowsky and Maksutow^[539], Palmirsky, and especially Nikanoroff^[540], took up this question, and communicated very encouraging results. Von Behring^[541] also found it very useful in certain cases. Thus, for the vaccination of guinea-pigs against tetanus toxin, he recommends the injection of a mixture containing antitoxin and an unneutralised excess of toxin. Under these conditions he easily succeeds in immunising these small animals in cases where all other methods fail. As a general method of vaccination against toxins, however, this method has not fulfilled its promise, and Roux, who tried it several times, was not at all satisfied with it.

This method of immunisation by mixtures of toxin and antitoxin is often spoken of as the method of vaccination by toxones. This name, “toxone,” was first applied by Ehrlich^[542] to a product developed by the diphtheria bacillus in culture media, a product less and differently toxic than is the true diphtheria toxin, yet capable of neutralising antitoxin. The idea of toxones presented itself to Ehrlich in connection with a fundamental fact noted by him, namely, that when to a non-toxic mixture of diphtheria toxin and antitoxin there is added one and even several lethal doses of the former, the animal is not affected. To make it succumb to intoxication it is sometimes necessary to add more than 20 lethal doses of toxin. To explain this paradoxical result, Ehrlich formulated the hypothesis that, in the soluble products of the diphtheria bacillus there exist two poisons: (1) the true toxin which exhibits a very strong affinity for antitoxin, and (2) the toxone which possesses less avidity for this antibody. When to an inactive mixture of the products of diphtheria bacilli and of antitoxin, there is added a fresh quantity of these same products, the added toxin, owing to its greater affinity, replaces the toxone of the previous combination. In the mixture to which is added one or several lethal doses of diphtheria poison, the toxone only is found free, all the toxin being combined with the antitoxin, and, as the toxone is only feebly toxic, the animal resists without suffering any serious illness.

Madsen^[543] adopted the theory of the diphtheria toxone, and affirmed that this substance poisons but slowly, produces neither early nervous symptoms nor loss of hair, but excites slight oedema at the point of inoculation and late paralyses. Susceptible animals may die from toxones, but very much later than as the result of poisoning by the toxins.

Ehrlich’s pupils have extended the theory of toxones to other bacterial poisons. Thus Madsen^[544] has described a similar toxone in tetanus poison—the tetanolysin of Ehrlich—which dissolves the red blood corpuscles, and Neisser and Wechsberg^[545] refer to a toxone in the poison produced by the staphylococcus.

Ehrlich also describes toxoids as occurring in diphtheria poison. The toxone, he maintains, is a product of the diphtheria bacillus itself, but the toxoids (protoxoids and syntoxoids) represent the toxin modified without further aid from the bacillus. The toxoids, though not toxic, retain all their avidity for antitoxin. According to Ehrlich’s conception, the molecule of toxin, under the influence of various factors, readily loses its toxic or toxophore group, capable of poisoning the animal, whilst still retaining its haptophore group, the group that combines with the antitoxin. The toxoids then would represent this haptophore group of the diphtheria toxin. Without being injurious to animals, the toxoids are capable of neutralising the antitoxin and of setting up in the animal the formation of this antibody. In the experiments carried out by the method of Babes and of the Russian authors we have just mentioned, there would be, according to the view held by Ehrlich and his school, an immunisation by the toxoids.

The toxones, however, are also capable of vaccinating against the toxin and the toxone and of giving rise to the production of a diphtheria antitoxin, active against these two poisons. This is what is affirmed by Madsen^[546] and by Dreyer^[547], according to a communication made by the latter to the International Congress of Medicine held at Paris.

By means of the various methods briefly described above, is obtained a real acquired immunity against the various bacterial and vegetable poisons and the venoms. On the other hand, with the methods of vaccination mentioned in the eighth chapter, which confer a substantial immunity against micro-organisms, we cannot demonstrate, in the vaccinated animals, a resistance against the corresponding toxins greater than in the unvaccinated control animals. The animals, so thoroughly vaccinated against certain micro-organisms that they withstood enormous doses of culture, did not become capable of resisting the minimal lethal dose of the poison. We are led to conclude, therefore, that immunity can only be obtained against certain of the toxins. For this reason we must regard the attempt made by von Behring to obtain a real immunisation against the toxin of cholera as an important forward step. Before von Behring’s attempt, various species of animals had been frequently and very substantially vaccinated against the cholera vibrio, but these animals, even when most thoroughly vaccinated, were completely non-resistant to the cholera toxin. Von Behring suggested to his pupil Ransom^[548] the idea of immunising guinea-pigs, not with microbial cultures living or dead, as had usually been done previously, but exclusively with the fluids of the cultures, deprived of the vibrios by filtration. In order, however, to attain the desired object, it was necessary to prepare fluids sufficiently active to poison the unvaccinated control guinea-pigs with certainty. The results of these investigations confirmed his anticipation, and Ransom soon found himself in possession of guinea-pigs well vaccinated against the cholera poison. He was mistaken, however, in supposing that, in all cases of immunity acquired against Koch’s vibrio, we have to do, in the main, with a purely antitoxic immunity. An investigation carried out in the Pasteur Institute^[549], whilst confirming the facts discovered by Ransom, lead to different results as regards their interpretation. It was demonstrated that the immunity against the vibrio is in no way founded on a resistance against its toxin and that we have to do with two very different acquired immunities. The vaccination obtained with the bodies of the micro-organisms induced a refractory condition against infection by the living vibrio, but not the slightest resistance against the toxin. The immunity, on the other hand, which is conferred by the injection of soluble products, deprived of the micro-organisms, is effective not only against the toxin of cholera, but also against infection by the vibrio. When an animal is vaccinated with cultures, or even with the bodies only of the vibrios, cholera toxin is introduced, but the toxin, under these conditions, is incapable of setting up antitoxic immunity. It would appear that the presence of the vibrios may constitute some obstacle to the production of this immunity.

Soon afterwards, Wassermann^[550] pointed out that the same rule applies in the case of the Bacillus pyocyaneus. With whole cultures of this bacillus he obtained in guinea-pigs an immunity exclusively against infection, whilst with cultures in a fluid medium, deprived of the bacilli, he was able to vaccinate his animals both against the pyocyanic toxin and against the infective peritonitis produced by the living micro-organism. The same double immunity could also be obtained in laboratory animals against the typhoid bacillus and several other bacteria.

When animals were subjected to different methods of vaccination against toxins, the manifestation of certain phenomena more or less constant was observed; amongst these must be pointed out especially the rise of temperature, a local reaction and certain modifications in the body fluids.

Fever is a very general symptom in the course of the vaccination of mammals. A rise of temperature is almost always observed as a result of the injection of toxins. It is very variable, both as regards duration and intensity, and cannot serve as an indicator of the result of the vaccination. In this respect, such great differences have been observed that the attempt to establish any general laws has had to be abandoned.

Local reaction is also a phenomenon which is very frequently observed during vaccination; to this von Behring^[551] paid great attention. He and his collaborators found that normal horses when injected subcutaneously with small or large doses of tetanus toxin did not present any exudation at the seat of inoculation. The horses which died as the result of a tetanus intoxication and those which got better behaved from this point of view in much the same fashion. In horses, however, which are being vaccinated and which are periodically subjected to gradually increasing doses of toxin, tumefaction at the seat of injection is never absent. Von Behring attributes this difference to the primordial insusceptibility of the living elements which govern exudation in the subcutaneous tissue to tetanus poison. It is only during the process of vaccination that these cells become susceptible and capable of manifesting a visible reaction. I consider that this difference is due more probably to a change in the chemiotaxis of the various elements which contribute to the inflammatory exudation reaction, from a negative to positive type. The cells do not react at the commencement, not because they are not susceptible to the toxin, but rather because their susceptibility is too great. During the course of vaccination they become sufficiently adapted to the poison to be able to manifest their normal inflammatory reaction. This explanation certainly harmonises with the fact that during the period of vaccinations in general and of vaccination against toxins in particular, the blood usually presents a more or less distinct hyperleucocytosis. Now, as is well known, this phenomenon of hyperleucocytosis is one of the most striking manifestations of a positive chemiotaxis in white corpuscles. It is true that, as to this reaction during the course of vaccination, the views of observers are not unanimous. Besredka^[552], as the outcome of his work on this subject, expresses himself very distinctly. “During the course of an immunisation against diphtheria toxin,” he writes, “one always observes a marked reaction in the goat, either at the beginning or at an advanced stage of the period of injections and especially in the first few hours after injection” (p. 322). Nicolas and Courmont^[553] in their first memoir maintain that hyperleucocytosis “is not necessary for immunisation.” Nevertheless, in the description of their experiments, which were performed on horses vaccinated against diphtheria, it is clear that the number of white corpuscles is often markedly increased. Further, in several cases they describe the formation of tumours at the point of inoculation, some of which end in suppuration. Under these conditions, it is not possible to deny a vaccinal reaction on the part of the leucocytes. Later, Nicolas, Courmont and Prat^[554] published a second memoir on the same subject, in which they seek to confirm their view of the uselessness of hyperleucocytosis in vaccination against the poison of diphtheria. They give details of experiments on several species of animals and insist specially on the conditions in which they have not observed hyperleucocytosis. “The doses from the first have always been extremely weak and with the addition of Lugol’s solution to attenuate them; only very gradually have we reached stronger doses, as that is one of the indispensable conditions for the avoidance of leucocytic variations, whilst obtaining a good and rapid immunisation” (p. 974). These special precautions to avoid hyperleucocytosis demonstrate clearly that this phenomenon is usually produced during the course of vaccination. It is quite natural that we should, by proceeding very slowly and with small doses of toxin, succeed in diminishing or even suppressing the afflux of leucocytes; but this fact cannot in any way minimise the importance of the leucocytic reaction in vaccination. In these particular cases, this reaction may take place without the number of leucocytes in the blood being noticeably increased. In reading the details of the experiments made by the Lyons observers, it will be seen that, in spite of all their precautions, they were unable to prevent the production of hyperleucocytosis. In all their cases, where they took the precaution to count the leucocytes several times a day, there was an undoubted increase of these cells. We may here recall Salomonsen and Madsen’s account of the immunisation of a horse against diphtheria toxin, in which they point out the frequency of tumefactions and even of abscesses. In most cases the pus was sterile, which renders it probable that the white corpuscles had accumulated at the seat of inoculation as the result of some influence exerted by the diphtheria toxin.

By far the most important and remarkable change met with in animals vaccinated against toxins and venoms, consists in the appearance of antitoxic power in their blood and fluids in general. This fact was, as already mentioned, first demonstrated by von Behring and Kitasato^[555] in the blood of rabbits immunised against tetanus. The blood itself, or the blood serum, mixed with a quantity of tetanus toxin more than sufficient to cause fatal poisoning, sets up no disease when injected into animals. In their earliest researches, von Behring and Kitasato kept the mixtures in contact in vitro for 24 hours, before injecting them into test animals. Later, they found that this prolonged contact outside the body was unnecessary and that they could obtain successful results by injecting the serum of vaccinated animals and the toxin simultaneously, even at different points of the body. This discovery was immediately afterwards applied by its authors to diphtheria and, in the case of both intoxications, confirmed by numerous observers.

For some time we were satisfied with vaccinating small laboratory animals and establishing the antitoxic power of their blood serum; later, the vaccination of large animals, especially horses, was commenced with the object of obtaining large quantities of antitetanus and antidiphtheria serum for medical use. During the course of these experiments the principal characters of the antitoxic fluids were established. It was deemed desirable to isolate the antitoxic substance from the blood serum in order to get rid of every unnecessary and inactive admixture, so that the antitoxin might be used in as pure a form as possible. This idea of isolating the antitoxic substance had, however, soon to be abandoned as impossible of realisation. Antitoxin is a non-crystallisable substance, of unknown chemical composition, which adheres firmly to the albuminoid substances of the serum. It is usually regarded as belonging to the same albuminoid group of substances, though it is not possible to prove this satisfactorily. Von Behring^[556], however, who studied this question in collaboration with Knorr, denies the albuminoid nature of tetanus antitoxin. After demonstrating that this antitoxin, when the antitetanus serum is submitted to dialysis, passes through the dialysing membrane, these observers found that they could not obtain the characteristic reactions of albuminoids in the dialysed fluid. It must be admitted, however, that this negative result is not sufficient to justify a denial of the albuminoid nature of antitoxin. When Nencki and Mme Sieber^[557] sought to produce the reactions of albuminoid substances with the digestive juice of Nepenthes (the well-known insectivorous plant) they got no result; but after the concentration of the juice in vacuo, it at once gave the characteristic reaction with nitric acid, and also with acetic acid, potassium ferrocyanide and Millon’s reagent.

The antitoxins may be precipitated along with the globulins and are distinguished, in general, by a fairly great resistance against physical and chemical influences. In this respect they are allied to the agglutinins, the fixatives and the precipitins, considered elsewhere, and are sharply distinguished from the cytases. The antitoxins resist temperatures which destroy the cytases and remain unaltered to beyond 60°–65° C. They are more stable than the delicate toxins of tetanus and diphtheria, but they are more easily altered than the toxins of cholera, of Bacillus pyocyaneus and the venoms. When stored in a dry state in the residue of evaporated serums and protected from light and air, the antitoxins will keep for a very long time without showing any notable attenuation. This property is very important in practice.

The antitoxins, in this respect also resembling the fixatives and the agglutinins, are humoral substances in the strictest sense of the term. They are found not only in prepared serums but abound also in the plasma of the circulating blood, and in the plasmas of the lymph and of exudations. Vaillard and Roux^[558] have shown that the clear acellular serous fluid of the oedema produced by the slowing of the circulation in rabbits vaccinated against tetanus toxin, is as antitoxic as the blood itself. Even the aqueous humour of a strongly immunised animal is antitoxic, though to a less degree. On the other hand, the saliva and urine exhibit very little antitoxic power, even when they are derived from animals hyperimmunised against tetanus toxin. Milk, as first demonstrated by Ehrlich^[559], is fairly rich in antitoxin, although much less so than the blood. According to the estimation of Ehrlich and Wassermann^[560], in the same immunised animal, milk contains one-fifteenth to one-thirtieth of the amount of diphtheria or tetanus antitoxin contained in the blood. Pus is always less antitoxic than blood or blood serum. According to Roux and Vaillard (l. c., p. 82), the pus of their rabbits vaccinated against tetanus toxin was only one-sixth or one-eighth as antitoxic as the serum of the blood. In Salomonsen and Madsen’s^[561] antidiphtheritic horse the cellular sediment of the pus was about one-half as antitoxic as the blood.

For the development of the antitoxic property in the fluids of the body, it is not essential that animals should belong to species susceptible to the corresponding toxin. Animals naturally most refractory against the poisons of diphtheria and tetanus are also capable of producing antitoxins. Vaillard^[562] demonstrated this fact in the fowl. This bird, which is naturally refractory against tetanus, usually acquires a very marked antitetanic power in its blood after one or more injections of tetanus toxin. He observed, however, that, in fowls thus treated, at a stage when the fluids of the body are antitoxic, the albumen of the egg is not so. The antitoxin, therefore, does not pass into this nutritive secretion, as it does into the milk of mammals. On the other hand, as has been demonstrated by F. Klemperer^[563], the vitellus of the eggs of fowls treated with tetanus toxin in time acquires an antitoxic property of the most marked character.

The antitoxins, found especially in the fluids of the body but only scantily in the cells, exert some action on the toxins. What is the nature of this action? This question, much studied and discussed, is one of very great importance in connection with the general problem of acquired immunity against toxins. In his first memoir, written in collaboration with Kitasato, von Behring (Deutsche med. Wchnschr., Leipzig, 1890, S. 1113) formulates his first thesis as follows: “the blood of a rabbit immunised against tetanus possesses the property of destroying tetanus toxin.” This idea of destruction, which would remove all toxic power from the poison, would naturally present itself to the mind and was at once accepted by a great many observers, but the numerous facts now accumulated on the subject will not allow us to accept a real destruction of toxins by antitoxins. Tizzoni^[564] was one of the first to point out certain contradictions between the theory of destruction and the phenomena produced in animals injected with tetanus toxin and antitoxin. Buchner^[565] also brought forward new facts which led him to conclude that antitoxin, instead of acting directly on the toxin, exerts its influence exclusively on the living elements, thus protecting the animal against intoxication. Amongst the arguments advanced by the Munich observer, the principal one is drawn from the different action of mixtures of tetanus toxin and antitetanus serum on various species of animals. It has been clearly shown that the guinea-pig is more susceptible to tetanus than is the mouse. In poisoning with tetanus toxin it requires an absolutely larger quantity of toxin to kill the guinea-pig than to kill the mouse. But if we take into account the weight of these animals, the conditions change completely. Thus, to cause a fatal tetanus in a guinea-pig, which weighs twenty times more than a mouse, we need only inject into the former a dose at most ten times greater than that necessary to produce fatal intoxication in the mouse. Buchner prepared a mixture of tetanus toxin and antitetanus serum which, in the mouse, produces no tetanic phenomenon or only sets up feeble and transient symptoms. According to the theory of direct action, we must assume that in this mixture the toxin is completely or almost completely neutralised by the antitoxin of the serum. But when Buchner injected the same quantity of mixture into guinea-pigs, without increasing it in proportion to the greater weight of these animals, he produced a tetanus of the most marked character. There has, consequently, remained in the mixture a sufficient amount of free toxin, whose tetanigenic action is manifested in the guinea-pig, an animal, as we have seen, more susceptible than the mouse. Buchner’s experiment has been verified by several observers. Roux and Vaillard^[566] carried out others which afford similar evidence. The same mixture of tetanus toxin and specific serum which is borne without the least difficulty by normal guinea-pigs, causes typical tetanus in other guinea-pigs of the same weight, and apparently in the best of health, but which have been immunised some time before against the Massowah vibrio. In another series of experiments, Roux and Vaillard injected into guinea-pigs a very large amount of antitetanus serum “capable of immunising them thousands of times,” and, shortly afterwards, a lethal dose of tetanus toxin. The normal guinea-pigs were thoroughly resistant to this test, whilst several guinea-pigs into which were also injected the products of other micro-organisms, acquired tetanus. Analogous results were obtained with mixtures of diphtheria toxin and antidiphtheria serum. Roux concludes from these facts “that the antitoxins act on the cells.” Against the theory of the destruction of toxins by antitoxins, he invokes the influence of heat on mixtures of these two substances. Calmette^[567], under Roux’s inspiration and in his laboratory, carried out various experiments on antivenomous serum. A mixture of this with snake venom, in such proportion that the poison became inactive, regained its toxicity after being heated for five minutes at 68° C. A normal animal, injected with this mixture, succumbed as if it had received pure venom. On being heated at 68° C. the serum lost all its antitoxic power over the venom, and the latter, which only becomes modified at a much higher temperature, remained intact. Later, a similar result was obtained by Wassermann^[568] in his experiments with pyocyanic toxin. This poison is resistant at even higher temperatures than is snake venom, whilst the antitoxin of the serum is destroyed under the same conditions as are the other antitoxins. Taking advantage of these peculiarities, Wassermann boiled the mixture of pyocyanic toxin and antitoxin serum, being careful to dilute it with two volumes of distilled water before doing so. This mixture which, before it was heated, was quite innocuous for guinea-pigs, again became a fatal poison after the destruction of the antitoxin.