X. RAPID AND TEMPORARY IMMUNITY AGAINST MICRO-ORGANISMS, CONFERRED BY SPECIFIC AND NORMAL SERUMS, OR BY OTHER SUBSTANCES, OR BY MICRO-ORGANISMS OTHER THAN THOSE AGAINST WHICH IT IS DESIRED TO PROTECT AN ANIMAL…

X. RAPID AND TEMPORARY IMMUNITY AGAINST MICRO-ORGANISMS, CONFERRED BY SPECIFIC AND NORMAL SERUMS, OR BY OTHER SUBSTANCES, OR BY MICRO-ORGANISMS OTHER THAN THOSE AGAINST WHICH IT IS DESIRED TO PROTECT AN ANIMAL

Immunity conferred by specific serums.—Analogy of the mechanism of this immunity with that observed in immunity obtained with pathogenic micro-organisms and their products.—Part played by phagocytosis in the immunity conferred by specific serums.—Influence of opium on the course of immunisation by these serums.—Stimulant action of specific serums.—Protective and stimulant action of normal serums.—Influence of fluids, other than serums: broth, urine, physiological saline solution, etc.

Antagonism between anthrax and certain bacteria.

[Sidenote: [315]]

We have seen how important in the study of acquired immunity against micro-organisms is the demonstration of the protective power of the body fluids. Without being absolutely general, this power is, nevertheless, widely diffused and is found even in certain examples of acquired immunity against micro-organisms belonging to the animal kingdom. Up to the present I have refrained from doing more than point out the presence, in the fluids of the immunised animal, of this protective property and have studied it exclusively in relation to the animal that produces it. We must now pass to the question: How do the protective substances act in the animal which receives them ready formed? This immunity, which has received from Ehrlich the name of passive immunity against micro-organisms, must now be examined.

[Sidenote: [316]]

The study we now propose to enter upon is rendered much easier from our study of the data acquired on the phenomena exhibited in the animal vaccinated with micro-organisms or their products, data already given in the preceding chapter. There is, indeed, a very striking analogy between the two kinds of immunity, and though we draw a sharp line of distinction between them, this is due to the fact that the immunity conferred by micro-organisms or their products requires some time for its development and endures for a long period, whilst the immunity due to the introduction of specific serums into an animal is set up immediately, but endures for a very short time only.

The diseases of the Invertebrata being seldom due to the micro-organisms that produce infections in the higher animals, the question as to whether the Invertebrata can be immunised by means of protective serums has not yet been studied. Still, we already have certain ideas on the protection of lower vertebrates by specific serums. Gheorghiewsky[464], in my laboratory, carried out some experiments on this point. He found that the serum of mammals (guinea-pig, goat) immunised against the Bacillus pyocyaneus, was under certain conditions capable of protecting the green frog against a dose of this organism that was always fatal to the control animals. When injected along with the Bacillus pyocyaneus, the serum did not prevent a fatal infection; sometimes this infection developed even more rapidly than in the control frogs. It was only when the protective injection was made 24 or, better still, 48 hours before the inoculation of the bacilli, that the protective action became evident. The serum used in these experiments was not bactericidal for the Bacillus pyocyaneus which grew most luxuriantly; but it agglutinated a large proportion of the bacilli. Gheorghiewsky pointed out, however, that frogs injected with cultures agglutinated by the goat’s serum died just as readily as did the control animals. As the phagocytic reaction was invariably very active in those frogs which resisted the virus, after the injection of protective serum, it is very probable that this fluid exercises a stimulant influence on the phagocytes.

[Sidenote: [317]]

This idea of stimulation by anti-infective serums in cases of temporary immunity conferred by these fluids, has already been set forth in my researches on the immunity of rabbits against the Gentilly cocco-bacillus, induced by the serum of vaccinated rabbits. This view, however, has not been favourably received, especially in view of the discovery of the phenomenon of the transformation of cholera vibrios into granules. Pfeiffer himself noted that this transformation took place not only in the peritoneal cavity of vaccinated guinea-pigs but also in the peritoneal cavity of normal guinea-pigs, into which he had injected small quantities of specific serum. As this latter fluid, in Pfeiffer’s hands, was incapable of transforming the vibrios into granules in vitro, he concluded that the cellular elements of the normal animal were endowed with the power of modifying the inactive substance of the specific serum into bactericidal substance. According to this conception the immunity conferred by this serum was not entirely passive since, in order to prepare the substance which transforms and kills the vibrios, the co-operation of the living cells was necessary.

My demonstration of the possibility of obtaining Pfeiffer’s phenomenon in vitro at once turned the balance in favour of the theory that the immunity induced by the specific serum is due to a direct humoral action upon the micro-organism. Under these conditions such immunity could only be interpreted as being purely passive. This view seemed to be finally established by Bordet’s discovery that a specific serum, inactive by itself, became capable of producing Pfeiffer’s phenomenon, as soon as a small quantity of normal, non-specific serum was added to it. Bordet[465] thus sums up his theory of the immunity conferred by specific serums: “Passive immunity is due, in part at least, to a chemical action exerted on the vibrios by two pre-formed substances, the one present in the animal before any injection is made, the other found in the serum that is injected; this phenomenon is purely chemical in the sense that it can go on without the aid of a vital reaction, of any new cell secretion: indeed it is found to take place in fluids from which the cells have been entirely removed” (p. 217). These demonstrations led up to the belief that the organism of the animal remained absolutely passive when it was subjected to the action of protective or anti-infective serums, and that the case of the cholera vibrio represented a kind of schema, which was applicable to the whole of the group of phenomena met with in passive immunity.

As in the study of the immunity obtained as the result of vaccinations with micro-organisms or their products, so in “passive immunity” there was seen only the direct chemical action of two substances on the micro-organism, and efforts were made to extend this demonstration to a series of infective diseases.

[Sidenote: [318]]

[Sidenote: [319]]

Pfeiffer and Kolle[466] having observed that the blood serum of persons convalescent from typhoid fever, as well as that of animals vaccinated with the typhoid bacillus, exhibited a great protective power for the guinea-pig, wished to get some idea of the mechanism of this immunity. They found that in the peritoneal cavity of guinea-pigs, inoculated with the typhoid cocco-bacillus and simultaneously subjected to the action of protective serums, the micro-organisms lose their mobility almost immediately. A little later, they exhibit a degeneration of form, become less refractile and disintegrate. After the injection of large doses of specific serum the bacilli, much as in the case of the cholera vibrio, become transformed into granules. “But,” say these authors, “this last mode of destruction, that is to say the formation of granules at the expense of the injected bacteria, does not occur with such remarkable regularity as it does in Pfeiffer’s phenomenon in the cholera vibrio” (p. 219). Whilst these changes are going on in the peritoneal fluid, the leucocytes begin to come up and to ingest the bacilli and their débris. “Phagocytosis, therefore, undoubtedly plays a part in the destruction of the bacteria. Nevertheless, as most of the injected bacteria die in the fluid of the exudation, phagocytosis can not be regarded as the cause of the protective action of the serum” (p. 220). We see from this description that even in the case of the typhoid cocco-bacillus the direct action of the fluids of the body is perceptibly less marked than in the case of the cholera vibrio. Even in the latter, however, it is necessary to make many reservations. The same laws apply to the immunity against this micro-organism, conferred by the serum of immunised animals, as to the immunity due to vaccinations by the vibrios or their products. To treat this subject fully one would have to repeat almost textually the two preceding chapters, but I will simply recall the fact that this transformation, almost general and very rapid, as we observed in vitro in vibrios placed in contact with fresh specific serum or with the mixture of this serum, heated to 55°–56° C., and normal unheated serum, is only met with in the animal body where phagolysis appears. Pfeiffer first observed the phenomenon which bears his name in the peritoneal cavity, and it is best seen in that situation during the period of the phagolysis of the white corpuscles. Vibrios, mixed with small doses of specific serum which by itself is able to render them motionless and agglutinate them, but which is absolutely unable to transform them into granules, present this transformation immediately they are introduced into the peritoneal cavity of normal guinea-pigs. In this case the vibrios, permeated by the fixative of the specific serum, are affected by the microcytase which has escaped from the injured phagocytes and is found in the peritoneal fluid. The preparation of the peritoneal cavity of normal guinea-pigs by means of an injection of broth or physiological saline solution the day before, prevents the production of Pfeiffer’s phenomenon, in spite of the protective serum, just as in vaccinated guinea-pigs. In both cases the vibrios, without being transformed into granules by the fluid part of the peritoneal exudation, are ingested by the phagocytes en masse and with extraordinary rapidity. This experiment was repeated by Garnier[467] with the typhoid cocco-bacillus. He first injected into the peritoneal cavity of young guinea-pigs several c.c. of physiological salt solution, of fresh broth or of some other fluid. The next day he introduced into the same situation typhoid cocco-bacilli mixed with blood serum from a donkey that had been for a long time immunised against this organism. A few minutes (2–4) after this latter injection the leucocytes, whose phagolysis had been prevented by the previous day’s preparation, were found crammed with cocco-bacilli. Some of these bacilli, like those still free in the peritoneal fluid, retained their normal form, but a very large number of those ingested by the microphages were already transformed into granules. This experiment affords fresh confirmation of the hypothesis that the substance which transforms the cocco-bacilli or the vibrios into granules is the microcytase. In the phagocytes in their normal condition this microcytase is found in the microphages, but during phagolysis a portion of it escapes into the surrounding fluid. In the control experiments made by Garnier with young normal guinea-pigs not prepared by preliminary injection, the simultaneous injection of typhoid cocco-bacilli and specific donkey’s serum set up this attenuated and not very typical Pfeiffer’s phenomenon described in Pfeiffer and Kolle’s memoir.

[Sidenote: [320]]

Soon after the discovery of Pfeiffer’s phenomenon I[468] was able to bring forward a proof that it was produced neither in the subcutaneous tissue, in the oedemas set up by the arrest of the circulation, nor in the anterior chamber of the eye of animals when cholera vibrios mixed with anti-infective specific serum were injected into these situations. Here the micro-organisms retain their normal form, remain quite alive and in this condition are ingested by the leucocytes which are brought up to the points invaded. These cells, attracted by the vibrionic products, do not undergo any phagolysis and, untrammelled, fulfil their phagocytic function. Inside them are found vibrios which have kept their elongated form and others which have become transformed into granules. The exudations containing these elements still give cholera cultures on nutrient media, a proof that some at least of the intracellular vibrios are alive. Here we have no destruction of the micro-organisms in the fluids of the body, consequently no direct action of the bactericidal substance. This substance, enclosed in the phagocytes, can only act through the intervention of these elements.

[Sidenote: [321]]

Mesnil[469] made analogous experiments with the Massowah vibrio, which, unlike the cholera vibrio, is peculiarly virulent when injected subcutaneously into guinea-pigs. In spite of this difference, this micro-organism, when injected along with protective serum into normal animals, behaves much as does the cholera vibrio proper. Mesnil injected the Massowah vibrios at the same time as the anti-infective specific serum, into the subcutaneous tissue of young and adult guinea-pigs and of young rabbits. In every case he observed the same reaction phenomena in the animal organism. The vibrios caused the formation of oedema at the point of inoculation and remained isolated in the fluid. The majority of these micro-organisms became motionless, but a few remained motile. Pfeiffer’s phenomenon was never observed. There was sometimes an aggregation of the vibrios, but this was not comparable with the marked agglutination brought about by the specific serum in vitro. The vibrios retained their power of reproduction, and Mesnil was able to observe them in all phases of division. Some hours (6–8) after inoculation the leucocytes began to come up to the seat of injection and set to work at once to ingest the vibrios. This phagocytosis became more and more marked, and finally there was ingestion of the whole of the micro-organisms. Drops of the exudation containing only intraphagocytic vibrios, when placed in the incubator, gave abundant cultures. The leucocytes died outside the animal body, whilst the vibrios continued to live and grow well under the new conditions. Certain leucocytes became three times their original size, and their contents were seen to be made up of vibrios closely packed together. The subcutaneous exudation, when withdrawn even eight days after the injection of the micro-organisms and sown on nutrient media, still gave colonies of vibrios.

It is evident, therefore, that the direct action of the protective serum on the vibrios was reduced to a mere trifle. It rendered them motionless and brought about a very slight clumping, but it was incapable of transforming the vibrios into granules or of destroying them. We see, then, that even in the case of the vibrios, the part played by Pfeiffer’s phenomenon is very limited. The destruction of the vibrios is effected with certainty, and completely, under the influence of the specific serums, not by a direct action of the two antibacterial substances but through the mediation of the phagocytes. Before the fixative, introduced with the protective serum, can bring about this result, the leucocytes, impressed with a special sensitiveness, must come up to the seat of inoculation, seize the micro-organisms and secrete around them their cytase. It is only as a result of these actions, purely vital, that the chemical or physico-chemical reaction of the substances which intervene in the destruction of the vibrios is brought about.

[Sidenote: [322]]

Under these conditions it can easily be understood that if the vital action of the phagocytes is retarded or depressed the injection of protective serum cannot preserve the life of the animal. Cantacuzène[470], who had already made a similar demonstration on guinea-pigs vaccinated against the cholera vibrio by these organisms or by their products, carried out numerous experiments on the action of opium on normal guinea-pigs simultaneously inoculated with vibrios and specific serum. Before injecting this mixture Cantacuzène narcotised his animals by means of tincture of opium. The great majority (⅘) of the guinea-pigs so treated died at the end of one or several days. The transformation of the vibrios into granules, under the influence of the serum, took place in the peritoneal cavity, but the leucocytes, on account of the narcotic action of the opium, were tardy in coming up. On their arrival in the peritoneal cavity they were capable of ingesting the granules, but absolutely refused to seize entire vibrios, always fairly numerous in the exudations. In spite of the appearance of a large number of leucocytes, these cells were still too weak to offer any adequate opposition to the vibrios, which increased in number and continued to multiply up to the death of the animal, when the exudation simply swarmed with very motile vibrios. Sometimes the struggle was prolonged. The weakened leucocytes allow the vibrios to develop, but, after a greater or less length of time, they regain their strength and begin to ingest the micro-organisms vigorously. Complete phagolysis follows, but the guinea-pig, attacked by the toxic products of the vibrio, finally succumbs in spite of the absence of free vibrios from its body.

An analysis of the phenomena observed in the body of an animal treated with antivibrionic serum, demonstrates that, in spite of a certain direct action of the substances contained in this fluid, there still remain a whole series of processes, amongst which the carriers of the cytases, that is to say the phagocytes, fill the most important rôle. Nevertheless, the cholera vibrio with its allied forms is still the most sensitive of all the micro-organisms to the bactericidal action of the fluids of the body. It may, therefore, readily be conceived that the more resistant micro-organisms are even less subject to the direct influence of the specific serums. Thus we have seen that the coccobacillus of typhoid fever presents, in the phagolysed peritoneal fluid, merely an attenuated form of Pfeiffer’s phenomenon. The other representatives of the group of bacilli are still less subject to the direct action of the serums, and Gheorghiewsky[471], in his studies on the Bacillus pyocyaneus, found that normal guinea-pigs, injected subcutaneously with anti-infective specific serum, and inoculated into the peritoneal cavity with this organism, present the same phenomena as those described in Chapter VIII. He never noticed either lysis of the bacteria in the fluids of the animal or their total transformation into agglutinated masses outside the phagocytes. The resistance offered by the animal was always in direct relation to the rapidity of the appearance and extent of the phagocytic reaction.

[Sidenote: [323]]

In order to determine the relative importance of each of the factors which act in the preservation of animals subjected to the influence of the specific serum, Gheorghiewsky repeated Cantacuzène’s experiments on the effect of narcotisation by tincture of opium. This alkaloid retards diapedesis, but does not affect the tactile sensibility or the motility of the leucocytes. The humoral properties, on the other hand, are not in the least affected by the narcosis. In spite of the fact that in guinea-pigs, narcotised and treated with anti-infective serum, the direct action was not interfered with, the animals always died because the retarded and incomplete phagocytic reaction was insufficient to destroy the bacilli rapidly enough.

Mesnil[472] studied the action of the specific serum against swine erysipelas on normal animals into which he had injected it some time before inoculation of the corresponding bacillus into the peritoneal cavity. This serum exercises a most marked protective action on the mouse, an animal very susceptible to the pathogenic action of this micro-organism. In mice so prepared complete and rapid phagocytosis takes place. These micro-organisms before being ingested by the phagocytes show no appreciable change; they are always stained very uniformly and intensely by Gram’s method, and they never swell up. The bacilli undergo no agglutination in the body of the mouse, a fact of which we can convince ourselves by examining hanging drops of the exudation. The phenomenon which strikes the observer most is the very pronounced phagocytosis, due principally to the activity of the microphages. Some hours after inoculation these cells are found to be crammed with bacilli, a large number of which no longer stain in the normal fashion. Without being transformed into granules, these micro-organisms undergo intracellular digestion which at the end of a few days is complete. This destruction is more rapid and complete in the microphages, slower in the macrophages. Drops of exudation collected from these mice, at a stage when the ingestion is completed, produce fatal septicaemia in untreated mice. This is proof that at the moment when they were seized by the phagocytes the bacilli still retained their virulence. Mesnil, as the result of his experiments, concludes that “the effect of the serum is to stimulate the phagocytes and especially the polynuclear forms; they ingest more quickly, they digest more quickly. The serum is, therefore, a stimulant of the cells charged with the defence of the animal” (p. 496).

[Sidenote: [324]]

We need not describe the phenomena produced in mice inoculated subcutaneously and treated with protective serum, for even in the peritoneal cavity neither Pfeiffer’s phenomenon nor any extracellular destruction of the bacilli can be observed. The micro-organisms, when subjected to the influence of the specific serum, readily absorb the fixative, as demonstrated by Bordet and Gengou[473]. This absorption must certainly favour the action of the intraphagocytic cytases. It is not, however, sufficient to explain the protective, anti-infective action of the serum. Such explanation was given by the experiments which Gengou, at my request, was good enough to make. He inoculated mice with the bacilli of swine erysipelas, mixed with specific serum heated to 55° C., to which was added some normal guinea-pig’s serum. The mice so treated resisted the infection but controls died in a few days. Being thus assured of the protective action of the serum, Gengou prepared the same mixtures of swine erysipelas bacilli and of the two serums; but, instead of injecting the whole of the mixture, he removed the bacilli from the serums, after a prolonged contact, and injected the bacilli alone into the mice. The bacilli had become permeated with fixatives, but, in spite of this, they killed the mice almost as quickly as the controls. Consequently, it is not the fixative adherent to the micro-organisms which determines the protective action of the specific serum. This fluid must contain another substance, one that will stimulate the phagocytes.

[Sidenote: [325]]

The analysis of the mechanism of the immunity termed passive, that is to say, communicated to normal animals by the introduction of an anti-infective specific serum, teaches us that, even when the direct action of the humoral substances is very limited, the protective effect, thanks to the stimulant action which brings about the destruction of the micro-organisms through the mediation of the phagocytic reaction, is still produced. The result at which we have thus arrived is confirmed by the examination of the phenomena observed in animals subjected to the action of anti-anthrax serum. Marchoux[474] first supplied us with precise details as to the mode of action on the rabbit of the serum of animals treated with anthrax bacilli. He found that, in the peritoneal cavity of rabbits injected the day before with anti-anthrax serum, the inoculated anthrax bacilli almost immediately become the prey of phagocytes. Within a couple of minutes after the introduction of bacilli into the peritoneal cavity, the great majority of them are ingested by the leucocytes; ten minutes later, there are no free bacilli. Not only the ingestion but also the destruction of these micro-organisms takes place with great rapidity, and even a few hours after the injection, the peritoneal exudation, when sown on nutrient media, remains sterile. In the subcutaneous tissue the phagocytic reaction requires a longer time than in the peritoneal cavity, nevertheless, it goes on very rapidly. Thus, when inoculated into the subcutaneous tissue of the ear of rabbits treated with specific serum, the bacilli are in great part ingested at the end of half-an-hour. At the end of an hour phagocytosis is usually complete.

In Marchoux’s experiments the importance of the part played by the phagocytes becomes still more prominent when it impedes their function in any way. Rabbits injected with anti-anthrax blood and 24 hours later inoculated below the skin of the ear with anthrax bacilli always resist infection, exhibiting the well-marked phagocytosis just mentioned. In other rabbits, however, prepared in the same way with the serum, but inoculated the following day into an ecchymosis excited by tapping the ear lightly, a certain number of the bacilli escape the phagocytes and succeed in setting up an abundant oedema followed by a fatal anthrax at the end of a few days. On making a post-mortem examination of these animals the bacilli were not numerous, but they were found in all the organs. The same result was obtained in another experiment in which Marchoux inoculated subcutaneously with anthrax blood which coagulated in situ rabbits prepared with specific serum. The blood clot attracted only the macrophages, as pointed out in Chapter IV. The microphages did not come up until late and then in small numbers. Now, as these are the phagocytes that are chiefly instrumental in destroying the anthrax bacillus, their absence allowed the bacilli to multiply and to set up a fatal anthrax. The rabbits prepared with the same serum but injected with anthrax blood diluted with broth (which prevents the formation of clot) completely resisted infection, thanks to the phagocytic reaction which went on without hindrance.

[Sidenote: [326]]

Sclavo[475] also, who made numerous investigations on the action of the anti-anthrax serum, is of opinion that this action is not a direct one upon the bacillus but is produced indirectly through the action of the animal organism. He maintains that the serum stimulates the function of the phagocytes and augments the bactericidal action of the body fluids. But since this bactericidal power enters the cytase as a substance destroying the micro-organisms, and this cytase is contained in the phagocytes, we can readily understand what a dominant part in the process these elements play.

Sobernheim[476], also, has paid much attention to the question now under discussion. As the result of his researches he comes to the conclusion that the anti-anthrax serum “cannot exert any effect on the virus by a direct action of the protective specific substances.” In order that the serum may be effective, the active intervention of the organism of the animal is necessary, otherwise, it is impossible to explain why the serum, used in the same proportion against the same quantity of anthrax bacilli, should protect one species of animals (the rabbit) and allow another (guinea-pig, mouse) to succumb. When Sobernheim tried to apply to anthrax the discovery of the transformation of cholera vibrios into granules, he got only negative results. There was nothing produced comparable to Pfeiffer’s phenomenon and the anthrax bacilli usually underwent no apparent modification. Sobernheim affirms also that the rapid phagocytosis under the influence of the serum, described by Marchoux, “does not appear to be produced under all circumstances” (p. 117). As, however, his researches on this subject were made on guinea-pigs which, in spite of the treatment with specific serum, always ended by succumbing to anthrax, we readily understand that his results cannot be compared with those obtained by Marchoux. I was present at the experiments of this observer and convinced myself of the accuracy of the facts recorded in his memoir.

[Sidenote: [327]]

Most of the examples here studied justify fully the hypothesis of the stimulant action of protective serums, a view that I formulated as the result of my researches on the immunity of rabbits against the Gentilly cocco-bacillus[477]. In this the first case of anti-infective immunity, due to the serum elaborated by an immunised animal, I could not find either a bactericidal action, however slight, or any agglutinative or attenuating property of the fluids of the body. As, on the other hand, this serum had no antitoxic power, everything indicated that we must look for its action, which was nil or very slight on the micro-organism, as being exerted on the organism of the animal into which it was injected for protective purposes. A comparative examination of the course of the phenomena in the subcutaneous tissue of the ear in rabbits, some of which received an injection of the specific serum into the veins whilst others were kept as controls, at once showed how widely different were the two cases. In the control animals, the cocco-bacilli immediately began to multiply without meeting with any opposition on the part of the organism of the animal; on the other hand, in the rabbits treated with serum, the serum became rich in leucocytes which at once set to work to ingest the micro-organisms. In course of time the latter gradually diminished in numbers, whilst the leucocytes went on increasing. The phagocytosis, also, became more and more marked. This struggle was continued for more than 24 hours, after which the purulent exudation, containing masses of leucocytes, no longer included any cocco-bacilli visible under the microscope either outside or inside cells. Nevertheless, this pus was still capable of producing a fatal septicaemia in untreated rabbits, clearly proving that it still contained some living and virulent micro-organisms. These cocco-bacilli persisted for a long time inside the phagocytes; their presence being demonstrated by injecting the exudation into unprotected rabbits and thus setting up a fatal infection. Finally, however, they disappear completely. On consideration of such facts as these I considered that I was justified in formulating the following conclusion at the end of my memoir: “From the facts I have described, taken collectively, we may draw the conclusion that the preservation of unvaccinated rabbits treated with serum is due to a superactivity of the phagocytic defence; and it is allowable to express the opinion that the protective serum of hog cholera acts in rabbits by stimulating the phagocytes, rendering them less sensitive to the toxins, and by stimulating them in their struggle against the bacteria” (p. 310). The facts since collected by various observers fully justify this hypothesis. Amongst the other micro-organisms against which a rapid immunisation has been obtained by means of serum, we must cite the cocco-bacillus of bubonic plague. Numerous experiments, carried out on several species of animals, have shown that antiplague serum markedly augments the phagocytic reaction.

[Sidenote: [328]]

In the group of the cocci, the streptococci have been specially fully studied from the point of view now under discussion. As already stated in another chapter, success has been attained not only in thoroughly immunising several species of animals against this dreaded micro-organism but active serums have been obtained capable of conferring distinct and certain immunity. The protective action of Marmorek’s serum, prepared at the Pasteur Institute, has been specially carefully studied. This serum is obtained from horses that have received numerous injections of various races of streptococci pathogenic for animals and for man[478]. At Louvain, Denys and his pupils prepared several other antistreptococcic serums and studied their protective effect on laboratory animals.

[Sidenote: [329]]

In collaboration with Leclef, Denys[479] began by vaccinating rabbits against streptococci and studied the mechanism of the immunity obtained in these animals. A summary of their researches will be found in the eighth chapter. Denys and Leclef considered that the serum of vaccinated rabbits intervenes in two ways, first by directly hindering the multiplication of the streptococcus and then by exalting the activity of the leucocytes. They applied these results to the case in which immunity is conferred upon normal rabbits by the intervention of the serum of the vaccinated rabbit, but they were unable to furnish any data bearing directly on this immunity. Somewhat later, Denys[480], in collaboration with Marchand, published another memoir in which he describes experiments on the mechanism of the immunity conferred on rabbits by injections of the blood-serum of vaccinated horses. From these experiments they draw the conclusion that “the serum of the horse immunised against the streptococcus possesses no bactericidal properties, properly so called, against this micro-organism; it does not affect it directly; but it contains a substance which renders the phagocytic power of the leucocytes extremely active. Even in the presence of small quantities of this serum, the white corpuscles rapidly ingest the streptococci and are capable of stopping all development so long as they retain their amoeboid movements.” “The action of the serum upon the leucocyte in its conflict with the streptococcus, is really derived from the horse immunised against this organism. It exists neither in the ordinary horse nor in the horse vaccinated against diphtheria” (p. 15). Against these experiments of Denys and Marchand we might bring the same objection that we raised against the analogous experiments of Denys and Leclef, because, in both cases, these writers lay too much stress on the presence or absence of the phenomena of phagocytosis in preparations kept outside the body of the animal. Under these conditions phagocytosis is effected in a fashion too artificial to be capable of furnishing exact information.

Von Lingelsheim[481] met Denys and Marchand with the fact that, in their researches, the serum of the horse immunised against the streptococcus was only feebly bactericidal. After a prolonged contact (6–12 hours) with a specific serum, the streptococci, when transferred to rabbit’s blood, showed retarded development as compared with streptococci subjected to the influence of the antidiphtheritic and antitetanic horse serum. Von Lingelsheim himself, however, points out that the bactericidal action of the antistreptococcic serum was feeble and transient, and required the intervention of the reaction of the animal cells within the body.

[Sidenote: [330]]

The researches carried out by Bordet[482] in my laboratory are not open to the objections that we were justified in putting forward against the experiments made by Denys and Marchand, since he carefully watched the phenomena of immunity as they developed in the body of the animal subjected to the action of antistreptococcic horse serum. Bordet began by studying the properties of this serum and accepted Denys’ and Marchand’s statement that bactericidal power, however small, was absent. The streptococcus grows as well in this serum as it does in that of the untreated horse. In the specific serum, however, markedly longer chains are produced than in normal serum. This difference is found only in the earliest period of the growth. The agglutinative power of the antistreptococcic serum is but feebly marked. The injection of a large quantity of this serum into a normal rabbit confers no bactericidal power upon the serum of this animal. “The serum extracted 24 hours after injection is quite as suitable for use as a culture medium as that furnished by the blood before the introduction of the serum. In both the micro-organism grows rapidly and vigorously” (p. 195). Consequently, in the antistreptococcic serum there is nothing comparable to what we obtain so readily with antivibrionic serum: nothing which recalls Pfeiffer’s phenomenon, even of an attenuated nature. We have already noted the result obtained by Bordet, according to which the streptococci, developed in the specific horse serum, were found to be endowed with their normal high virulence. The antistreptococcic serum, injected into the peritoneal cavity of the rabbit the day previous to the microbial inoculation, protects the animal absolutely, provided that the micro-organisms be not too numerous or the quantity of serum not too small. Under these conditions the virus is ingested pretty rapidly and, so far as we know at present, completely. The micro-organism is thus prevented from developing and the animal remains in good health, whilst the control animal, which has received no serum, dies in a very short time.

When the number of streptococci is increased the effort of the animal organism to get rid of them becomes, in spite of the protective serum, more severe and much more prolonged. Some of the micro-organisms certainly become the prey of phagocytes, but a sufficient number remain free in the peritoneal cavity to multiply rapidly. When the number of streptococci has become sufficiently great a phenomenon, to which Bordet gives the name of “phagocytic crisis,” is suddenly observed. In the peritoneal exudation, which has become thick and has taken on the aspect of a homogeneous and white pus, a most rapid phagocytosis is evidently set up. In a short time the whole of the streptococci, which were swarming outside the cells, are ingested by the leucocytes. “The essential condition for recovery is always this complete ingestion” (p. 203). If the ingestion is not general, the rabbit may die, although much later than the control animal.

[Sidenote: [331]]

The phases of the struggle between the animal organism, when subjected to the influence of the protective serum, and the streptococcus, recall Salimbeni’s experiments on immunised horses. The rabbit, in which phagocytosis could not take place at once owing to the presence of too large a number of micro-organisms, exhibits first a stage of free development of the streptococci, after which the phagocytes begin to fulfil their antibacterial function. Here it is especially the macrophages which act; the microphages, although present in fairly large numbers, are entirely inactive. This first stage of phagocytic reaction is insufficient. It is followed by a period when the streptococcus appears to gain the upper hand. Many small chains, having escaped the phagocytes, multiply and give birth to quite a new generation of micro-organisms. If a fresh impulse to phagocytosis does not take place the animal dies from infection. When, however, the protective serum has been of sufficient strength, a new army of leucocytes arrives on the scene and these become masters of the situation. Phagocytosis becomes complete and microphages as well as macrophages devour a large number of streptococci.

Bordet, who, through his previous investigations, was well acquainted with the direct action of the protective serum on vibrios, could find nothing resembling it taking any part in the struggle of the organism of the animal treated with antistreptococcic serum against the streptococcus. The most that he could find was that the streptococci which again begin to swarm in the exudation are smaller in size than the normal streptococcus. It must be accepted, as indicated by the most recent researches, that this micro-organism becomes permeated by the fixative substance of the specific serum. We know already, however, that this fixation cannot deprive the micro-organisms of their virulence. In any case, then, a large share in the process must be attributed to the action of the phagocytes, stimulated by the protective serum, in the struggle of the animal against the streptococcus.

[Sidenote: [332]]

Having considered this series of examples of immunity against bacteria conferred by specific serums, we are in a position to form some idea of the mechanism of this immunity. Before we come to any general conclusion, it may be useful to glance at an example of this so-called passive immunity against a micro-organism belonging to the animal kingdom. Such examples are not numerous, as, in the majority of cases of acquired immunity against Protozoan parasites, the serum is inactive and incapable of communicating immunity to normal individuals. We have only a single example, the Trypanosoma of rats, against which Dr Lydia Rabinowitch and Dr Kempner[483] have demonstrated the possibility of immunisation by the blood serum of vaccinated white rats. The mechanism of this immunity has been studied by Laveran and Mesnil[484], who found that it was like that described (Chap. VIII) in connection with the immunity in white rats, conferred by the inoculation of living Trypanosomata. The specific serum does not affect these infusoria except that it brings about slight agglutination. Trypanosomata placed in contact with it retain their pristine vitality and motility. This fact led Mme Rabinowitch and Dr Kempner to advance the hypothesis that the protective action of the serum must depend upon its antitoxic power. Since, however, in the infection of rats by the Trypanosomata, the toxic action is very feeble if not nil, it is very difficult to accept this view. It certainly appears to be much more probable that the serum acts in this case, as in many others, by stimulating the phagocytic reaction. The rapidity with which the living Trypanosomata are ingested by the phagocytes has been shown by Laveran and Mesnil.

[Sidenote: [333]]

Reviewing the whole of the data on immunity produced under the influence of anti-infective or protective serums, it is evident that they fall under two main categories. On the one hand there is a direct action of these serums on the micro-organisms, an action that is either microbicidal properly so called, agglutinative, or fixative. On the other hand, a stimulation of the phagocytic defence which leads to the final destruction of the micro-organisms is set up. This last factor is general; even in the case where the direct action is most marked (vibrios in the phagolysed peritoneal cavity), its importance is considerable. The micro-organisms which can be deeply injured by the direct action of the specific serum are few in number. In most cases this action is a feeble one and needs, for its completion, effective co-operation on the part of the phagocytes. In this respect micro-organisms present a whole gamut which begins with the cholera vibrio, the micro-organism most sensitive to the action of the body fluids, and ends with the Trypanosoma of the rat, a flagellated Infusorian which cannot have even its motility affected by the direct action of the fluid elements. In all these cases, of course, the immunity conferred by the serums is due to the final destruction of the micro-organisms which invariably resolves itself into the same fundamental act—digestion by the cytases, a phenomenon which can only be produced at all quickly by the action of cytases contained in the protective serums or that have escaped from the phagocytes during phagolysis. The digestion by the cytases may also, and this is usually the case, be effected only after the manifestation of a regular series of vital phenomena on the part of the defensive elements of the body. As this factor fills such an important rôle, it is readily understood that we can not accept the term passive immunity by which to designate the immunity conferred by the specific serums. The action of the cytases, which is necessary to bring about the final result in this immunity, depends too much on the activity of the cells which contain the bactericidal ferment. For this reason, when the functional activity of the phagocytes is in abeyance or is retarded, the animal succumbs, in spite of the presence in its organism of a more than sufficient quantity of cytases. In this connection Wassermann’s[485] suggestion of adding normal serums rich in cytases to the specific serums must be regarded as very apposite. When protective serums poor in cytases or which have lost them as the result of heating, of the use of antiseptics, or simply from the influence of time, are injected, no immunising effect is ever obtained, simply because of the inactivity of the phagocytes, the cells in which the cytases are found. If at the same time normal serum rich in cytases ready prepared be injected, better results should be obtained. We may recall here an analogous example—the anthrax of the rat. Although possessing a large quantity of cytase, very effective against the bacillus, the organism of the rat can make no use of it, because the phagocytes which contain it do not manifest a sufficient activity. But the injection into a rat of blood serum from the same species containing a certain amount of cytase that has escaped during the formation of the clot, is sufficient to preserve the animal against a fatal infection.

[Sidenote: [334]]

To support his view, sound in principle, Wassermann made an experiment the interpretation of which presents certain difficulties. He injected guinea-pigs with protective antityphoid serum, in a dose insufficient to protect them against a fatal infection. By introducing along with this serum a certain quantity of normal ox serum which, by itself, is also incapable of averting a fatal issue, Wassermann obtains an absolute immunity of his animals. This immunity is due, according to Wassermann, to the cytase of the ox serum acting along with the fixative of the specific serum. The united action of the two ferments causes the death of the micro-organisms. Besredka[486] has justly observed that normal ox serum contains, in addition to cytases, a substance which exerts a distinct agglutinative action on the typhoid cocco-bacillus and another which stimulates the phagocytic action. These two substances resist a temperature of 55°–60° C., and Besredka shows that with normal ox serum, deprived of its cytases by heating as above, we can obtain the same protective effect as with the same serum unheated.

As the result of another series of experiments, Wassermann[487] recognises the immunising action of normal serum heated to 60° C. and so entirely deprived of its cytases. Into the peritoneal cavity of guinea-pigs he injects, mixed with heated normal rabbit’s serum, a dose of typhoid cocco-bacilli several times greater than the lethal dose. The guinea-pigs resist this completely. Analysing the mechanism of this immunity, Besredka (l.c. p. 229) attributes it to the combined action of the agglutinin and of the substance which stimulates the phagocytes. We have here another proof that the stimulins which play such an important part in immunity conferred by serums, are found not only in the specific serums, but also in normal serums, whether unheated or heated to 55°–60° C.

The protective property of the normal serums of man and animals against the cholera vibrio has already been referred to. We may now go a little more deeply into the mechanism by which these serums act. This task is an easy one thanks to the important work by Issaeff[488] carried out in R. Pfeiffer’s laboratory. Having confirmed the observation, made by other investigators, that blood serum from the human subject, whether in health or affected by any disease, is capable of protecting the guinea-pig against the cholera vibrio provided that it is injected 24 hours before the micro-organisms, Issaeff studied the phenomena observed in the peritoneal cavity of the animals experimented upon. By means of small capillary pipettes he drew off at intervals a small quantity of fluid from the peritoneal cavity and examined it in hanging drop or in stained preparations. Some time after the injection this fluid became more and more rich in leucocytes which seized the vibrios, ingested and destroyed them. To obtain this protective effect it was necessary to inject from 0·1 to 5 c.c. of human blood serum. With these doses he could prevent, not only infection of the guinea-pigs by the cholera vibrio, but also the lethal effects of other vibrios. The protective action of normal human serum is general, therefore, and not specific, such as is the immunity conferred by the serums of vaccinated animals or of the human subject who has suffered from an attack of cholera.

[Sidenote: [335]]

Shortly afterwards Funck[489] confirmed this result in the case of the typhoid cocco-bacillus. He observed that normal horse’s serum, injected as a protective agent in the dose of half a c.c. into the peritoneal cavity of the guinea-pig, preserved this animal from a fatal infection. Pfeiffer and Kolle and Chantemesse and Widal obtained the same results with human serum. The former observers lay special stress on the non-specific character of this protective action of normal serums. As to its mechanism, Funck sums it up as follows: “the specific serum brings about a rapid lysis of the bacilli, normal serum acts in a much more limited fashion; if the dose is very large and if the animal resists infection, the phenomena of extracellular degeneration are rarely appreciable, and it seems that here the specially important factor is the intracellular destruction of the bacteria, in the phagocytes” (p. 70).

Wassermann has shown the protective action of normal serum against the experimental disease produced by the staphylococcus. This action, although not absolutely general, is nevertheless widely distributed. Wassermann[490], from comparative investigations on this subject, came to the conclusion that “the serum of a different species of animal acts by greatly increasing the resistance, whilst the serum of the same species produces an effect which is not nearly so marked.” As in these normal serums a stimulating influence on the phagocytes is specially marked, it may readily be understood that the serum of the same animal or of the same species does not produce so energetic an effect as the serum of a different species. As these normal serums possess, not only the property of exciting phagocytosis, but often also that of rendering motionless and of agglutinating certain micro-organisms, there might be some difficulty in interpreting the part played by these serums. It may be useful, therefore, to pass in review the protective action of fluids less complicated than blood serums.

[Sidenote: [336]]

Issaeff, in the work already cited, demonstrated that not only normal serums but a whole series of fluids, such as urine, broth, etc., exert a protective effect against microbial infections. These fluids must be injected about 24 hours before the introduction of the bacteria. The best method consists in injecting them directly into the peritoneal cavity, after which the animals acquire an immunity against absolutely fatal doses of cholera vibrios. Funck verified this observation for the infection caused by the typhoid cocco-bacillus, and Bordet confirmed it for the streptococcus. The injection of peptonised broth into the peritoneal cavity of the normal guinea-pig, made 24 hours before an inoculation of double the fatal dose of the streptococcus, exerts a distinct protective action; the infection does not kill the animal. This broth is neither bactericidal, attenuating, nor agglutinative; it forms a good culture medium for the streptococcus and possesses no fixative power. Consequently it does not act directly on the vitality or virulence of the micro-organism; nevertheless, it is distinctly protective.

According to Issaeff’s researches, the protective substances used by him must be arranged in the following order as regards their action against the cholera vibrio. Tuberculin is the most effective; then comes a 2% solution of nuclein, followed by normal human serum, broth, and urine, whilst physiological saline solution is the least active. All prevent infection by the vibrios, but the protection is effective for some days only; this protective action is exerted against various kinds of bacteria, being in no sense specific.

Pfeiffer lays so much stress on the great difference between the protective power of normal serums, as well as of the other fluids mentioned, and that of the anti-infective specific serums, that he even proposes to classify the first group as giving rise to pseudo-immunity or resistance. This view is certainly an exaggerated one, because it is difficult to draw a very distinct line between the two groups of phenomena. There are normal serums, of which 0·1 c.c. is quite sufficient to confer the protective effect, just as there are specific serums of which it is necessary to make use of a much greater dose to attain the same result.

[Sidenote: [337]]

Protective fluids, other than the serums, only manifest their influence by exciting a great phagocytic “superactivity.” As the result of their injection into the peritoneal cavity of normal guinea-pigs, first a transitory phagolysis is induced, this being soon replaced by a very considerable afflux of leucocytes, which is maintained for 24 hours or longer, and then gives place to the normal condition. It is during the period of the greatest leucocytosis of the peritoneal fluid that the animal exhibits the most marked resistance against infective micro-organisms. The vibrios are rapidly ingested by the phagocytes, without having previously been acted upon by the “humours.” Bordet shows that the same thing happens in the case of the streptococcus inoculated into guinea-pigs after a protective injection of peptonised broth.

[Sidenote: [338]]

We have observed the same phenomenon in guinea-pigs and white rats inoculated with the cocco-bacillus of plague. Treated with freshly prepared peptonised broth the day previous to inoculation, these animals oppose to the micro-organism a much more marked resistance than do the control animals. The injection of the cocco-bacillus of plague sets up a marked phagocytosis on the part of the macrophages. These cells ingest large numbers of micro-organisms which, after a time, have all passed into the phagocytes. If a drop of the peritoneal exudation is now withdrawn, we find only intracellular cocco-bacilli (fig. 43). If the drop be kept for some time outside the animal and at a suitable temperature the macrophages may be seen to perish and the micro-organisms to develop in their contents. We thus obtain abundant cultures which pass from the interior of the macrophages into the fluid of the exudation (figs. 42, 44, 45). When the animals are not sufficiently protected the same phenomenon is observed in the peritoneal cavity of the living animal. The macrophages, crammed with cocco-bacilli, burst, allowing the micro-organisms to escape. These multiply in the peritoneal fluid and spread through the animal, which soon dies.

Wassermann affirms that “the artificially increased resistance is nothing but an active and reinforced afflux of the complements (cytases) towards one point in the animal, for the purpose of digestion.” (Ztschr. f. Hyg., Leipzig, 1901, Bd. XXXVII, S. 199.) Wassermann does not explain how this afflux of cytases is produced. The entirely concordant researches on this point by Issaeff, Funck, Bordet, and ourselves, prove that this afflux takes place not through the mediation of the fluids, but solely through the phagocytes, the carriers of the cytases. Consequently it is beyond dispute that in the immunity conferred by physiological saline solution, broth, and several other fluids, we have to do solely with an augmentation of the phagocytic reaction. In the immunity conferred by normal or specific serums, this same stimulating factor still plays the more important part. Along with it, however, there is an intervention more or less pronounced, according to circumstance, and more or less frequent, of cytases, brought by the serums prepared outside the body or that have escaped during phagolysis, as well as of substances truly humoral, such as the fixatives or the agglutinins.

[Sidenote: [339]]

Amongst the non-specific substances which are capable of conferring an immunity more or less stable, must be placed the products of micro-organisms other than those against which we wish to protect the animal. Pasteur[491] noted that when the anthrax bacillus, mixed with other micro-organisms, in themselves inoffensive, is inoculated into animals, anthrax does not develop and the animals remain well. Later, Emmerich[492] showed that the streptococcus of erysipelas exerts an antagonistic influence against the anthrax bacillus. He succeeded in immunising and even in curing rabbits inoculated with anthrax, by submitting them to the action of this streptococcus.

These experiments served as the starting-point for several works on the vaccination of animals against anthrax by means of various micro-organisms, as well as by their products. Pawlowsky[493], Watson-Cheyne[494], and Bouchard[495] have proved that bacteria not very pathogenic and even saprophytes, such as the Coccobacillus prodigiosus, Friedländer’s bacillus, and the Bacillus pyocyaneus, were also capable of preventing infection by the anthrax bacillus. Freudenreich[496] showed that not only did the bacillus of blue pus exert an antagonistic action but that the same effect could be obtained with sterilised cultures of this organism. Woodhead and Cartwright Wood[497] studied the vaccinating action of these products on rabbits inoculated with virulent anthrax bacilli. The animals resisted completely or survived for some time. Analysing the phenomena produced under such conditions, these two authors came to the conclusion that the action of sterilised cultures of Bacillus pyocyaneus is “indirect and as taking place either by opposing itself to the action of the poison upon the tissues, or by stimulating certain tissues and increasing their functional activity.” With the object of obtaining an exact interpretation of this antagonistic influence I suggested to M. Blagovestchensky[498] that he should investigate in detail the phenomena which take place in the organism of rabbits inoculated with the anthrax bacillus and submitted to the action of sterilised cultures of the Bacillus pyocyaneus. At the very outset this observer was met by the fact that these cultures act directly upon the vitality of the anthrax bacillus. Thus the association of the former with the anthrax bacillus in vitro was sufficient to interfere with the development of the latter. Under these conditions he had to renounce the investigation of the part played by the cellular elements of the rabbit in the antagonism of the two bacteria.

[Sidenote: [340]]

Friedländer’s bacillus has been found to be much more suitable for this line of research as is shown by work carried out by Freiherr von Dungern[499] in my laboratory. This observer convinced himself that “anthrax bacilli are weakened neither by the encapsuled bacilli nor by the substances which they contain.” These micro-organisms do not interfere in the slightest with the anthrax bacilli either outside or within the animal, and “when the anthrax infection does not become generalised it is due to the fact that the anthrax bacilli are ingested by the phagocytes at the seat of inoculation and destroyed within these cells” (p. 183).

In this action of foreign micro-organisms upon micro-organisms against which we wish to protect the animal we have to deal with something analogous to the condition we obtain when immunising with normal serums or with any other kind of fluid. In both cases immunity is rapidly established, but it is very transient and is confined to a stimulation of the phagocytic resistance. Direct action may also intervene, as in the case of Bacillus pyocyaneus, but it is not indispensable. The animal whose phagocytes are in a condition of superactivity can do without this direct action, its own resources being sufficient to arrest anthrax.

Following the same lines of investigation as those on the antagonism between the anthrax bacillus and several other micro-organisms, Klein[500] has demonstrated that, in order to prevent a guinea-pig from contracting experimental cholera peritonitis, it is only necessary to inject into it, the day before infection, a culture of Finkler and Prior’s vibrio or of certain other bacteria. These experiments by Klein served as the point of departure for Issaeff’s work which led to the discovery of the stimulating influence of all kinds of fluids injected into the peritoneal cavity of guinea-pigs.

In this transient immunity obtained with products foreign to the micro-organism against which one is vaccinating, the most constant and consequently most important part is again played by the phagocytes. But there is associated with it an influence, greater or less in degree, of substances present in the serums, such as the microcytases and fixatives, which are able to exercise a direct action on the pathogenic micro-organisms. In all cases known and analysed up to the present, the intervention of the living organism of the animal is indispensable, consequently this form of acquired immunity against micro-organisms cannot be regarded as being really passive.

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XI. NATURAL IMMUNITY AGAINST TOXINS

Examples of natural immunity against toxins.—Immunity of spiders and scorpions against tetanus toxin.—Immunity of the scorpion against its own poison.—Antivenomous property of the blood of the scorpion.—Immunity against tetanus toxin in the larvae of Oryctes and in the cricket.—Immunity and susceptibility of frogs against this toxin.—Natural immunity of reptiles against tetanus toxin.—Antitetanic property of the blood of alligators.—Immunity of snakes against snake venom.—Immunity of the fowl against tetanus toxin.—Immunity of the hedgehog against poisons and venoms.—Immunity of the rat against diphtheria toxin.

[Sidenote: [341]]

As in this book we are dealing specially with the immunity against infective diseases, the question of the resistance of the animal to poisons interests us only in so far as it is related to immunity against micro-organisms. Consequently the reader must not expect a treatise on intoxications properly so called nor one on immunity against all kinds of poisons. To perform such a task we should have to far overstep the bounds of the subject that we have chosen and enter upon an examination of questions which are beyond our sphere. Our chief aim is to present to the reader a summary of our present knowledge on immunity against microbial toxins and to establish the relations between this kind of immunity and immunity against infective micro-organisms. In order to do this, however, we shall have now and again to go beyond the limits of our programme and discuss certain problems bearing on the resistance of the animal organism against poisons not of microbial origin.

[Sidenote: [342]]

The immunity against toxins, like that against the micro-organisms themselves, may be either natural or acquired. As many poisons have been known from time immemorial, we are able to collect numerous observations on the resistance of the animal organism to such substances made when there was no idea of immunity against infective diseases. The etiology of intoxications is often much more evident and simple than is that of infections; this is one of the reasons that the older conceptions on the subject of immunity against poisons were more advanced than were those on immunity against infective diseases.

Several examples of natural immunity in the lower animals have already been cited. Thus, we have seen in previous chapters that the Infusoria are resistant to poisons that exert a powerful action on a large number of the higher animals, such as the tetanus and diphtheria toxins and especially the ichthyotoxin of eel’s serum. We have mentioned the case of the larva of Oryctes nasicornis which is unaffected by large doses of the toxins of certain bacteria and which at the same time is very subject to fatal infections by very small doses of the bacteria that form the poisons. These larvae, like those of the cockchafer, are, however, fairly susceptible to the poison of the scorpion. Several other species of Arthropoda, which have been studied from the point of view of immunity against toxins, have exhibited analogous features. Thus spiders and scorpions are refractory to tetanus toxin. In one experiment I injected into the abdominal cavity of a Mygale from the Congo (which weighed 7 grm. 5) 1 c.c. of tetanus toxin on two several occasions. This dose is sufficient to kill, with the symptoms of tetanus, 1000 mice of double the weight. The spider, kept in the incubator at 36° C., remained quite well during the two months that the experiment lasted. It exhibited no symptom, not even transient, of muscular stiffening, nor any change in its habits and natural functions. The tetanus toxin disappeared from the blood of the Mygale, but this blood at no time showed the slightest antitoxic power against this poison. This example of natural immunity cannot, therefore, be ascribed to any antitoxic property of the fluids and must be regarded as a case of immunity of the tissues—von Behring’s histogenic immunity. In the present imperfect state of our knowledge it is impossible to describe precisely the mechanism of this immunity. When we say that the spider is refractory to the tetanus toxin because its susceptible elements have no receptors capable of seizing the haptophore group of this poison, we simply give expression to a hypothesis which we are not in a position to verify by experiment.

[Sidenote: [343]]

The scorpion, a well-known representative of the Arachnida with segmented abdomen, shares with the Mygale in the immunity against tetanus toxin. The Algerian and Tunisian scorpions (Scorpio afer and Androctonus occitanus) withstand the action of doses of this poison which are fatal for 1000 mice and more. Taking weight as our standard we may inject into them, with impunity, more than 5000 times as much toxin as into mice, without setting up a single morbid symptom. Scorpions, like the Mygale, live well in the incubator at 36° C., where they are kept whilst submitted to the action of the tetanus poison. Here again we have to do with a case of histogenic immunity. The fluids of the scorpion exert no antitoxic action. When blood from the normal scorpion is mixed with various doses of tetanus toxin and injected into mice these animals contract tetanus and die just as do the control animals. In certain exceptional cases some slight retardation was observed, but the blood of the scorpion is, in most cases, incapable of preventing tetanus in animals susceptible to this disease.

Scorpions, injected with tetanus toxin, do not retain it in their blood for long. A few days after the injection of the tetanus poison such blood, when injected subcutaneously into mice, excites no trace of tetanus. The preparation of extracts of the different organs of scorpions treated with tetanus toxin demonstrates that the liver and the liver only absorbs the poison. It is found there a few days after the injection of the toxin and it remains there unaltered for some considerable time. The exudation of the liver of scorpions, killed a month or more after the introduction of the toxin into the general cavity, injected into mice sets up a typical and fatal tetanus.

The presence of the tetanus toxin in the organism of scorpions does not give rise to the production of antitoxin. At any rate a whole series of experiments on this point carried out by us never gave a positive result. The scorpions resisted repeated doses of the tetanus toxin and lived without any difficulty at 36° C., but their blood was never at any period capable of preventing mice from contracting fatal tetanus. Nevertheless the scorpion may possess antitoxic power.

[Sidenote: [344]]

Everyone has heard of the supposed suicide of the scorpion. We are told that when this animal finds itself under conditions in which its death is inevitable, it stings itself with the end of its tail and dies from the effect of its own poison. A simple method of reproducing this experiment is actually described:—Surround the scorpion with a circle of fire. The animal rushes in all directions to find a way out, and finding none, deliberately commits suicide. Bourne[501] at Madras carefully investigated this question in a large species of Indian scorpion and demonstrated the absolute erroneousness of the story of suicide which, had it been true, would have afforded a unique example of voluntary death in animals. On carrying out the classic experiment he observed that within this ring of fire the scorpion is subjected to a very high temperature. When the temperature reaches 40° C. the scorpion begins to grow weak and as the temperature approaches 50° C. it passes into a comatose condition. Moreover Bourne showed that the scorpion’s poison, which is fatal for large spiders, insects, and vertebrates, was innocuous for individuals of the species furnishing it.

I can confirm all the statements of this English observer. When I was studying the embryology of the scorpion I repeatedly tried the experiment but the animal never committed suicide. Further, I repeatedly assured myself of the innocuousness of the scorpion’s poison when injected into individuals of the same species, and I was able to demonstrate most conclusively that the blood of the scorpion is endowed with undoubted antitoxic power. The addition of 0·1 c.c. of this blood to a dose of poison which kills mice in half-an-hour is sufficient to enable a mouse injected with the mixture to resist it completely. This antitoxic power is the same in the Scorpio afer and in the Algerian Androctonus. An emulsion of the liver of the scorpion, however, is absolutely incapable of preventing fatal intoxication of mice.

[Sidenote: [345]]

This case of antitoxic action is the only one I have been able to demonstrate in an invertebrate. Must we regard it as a case of natural innate antivenomous power or as something acquired during the life of the animal? It is not easy to settle this question by experiment. We can certainly procure new-born scorpions and rear them for some time, but the quantity of blood that can be got from them is insufficient for injection for protective purposes. Scorpions do not love one another and when kept together we often find them engaged in fierce and mortal combat, the stronger killing the weaker and sucking their blood. It is therefore possible that, in some stage of their life, the scorpions find means of vaccinating themselves against their own poison either through the intestine or as the result of punctures caused by the point of the tail. It would be very interesting to study this question under favourable conditions, because it is capable of throwing light on the problem of the origin of antitoxins, from a general point of view. Whichever view be taken, the acquisition of any antitoxic property by the blood in the Invertebrata must take place slowly and with great difficulty as is shown by our want of success with tetanus toxin.

Insects are, as a rule, very tolerant of this latter poison. As, however, the tetanus toxin (we shall illustrate this later) only acts well and in small doses at a high temperature (about 30° C.) and as most insects do not readily adapt themselves to this temperature, it was necessary to choose species capable of living at these high temperatures and for this line of study the larva of Oryctes is most suited. It flourishes well at a temperature of 30°–36° C., and under these conditions exhibits a much greater resistance to infection by Isaria than at lower temperatures. It can be kept in the incubator for months if placed in glass jars filled with earth mixed with tanner’s bark. The injection of enormous quantities of very active tetanus toxin directly into the blood has not the slightest effect on these larvae. Whilst, however, the blood fluid of the Arachnida rapidly gets rid of the poison, that of Oryctes retains it for a very long period. If a small quantity of blood be taken from larvae several months after injection and then injected into mice, these animals contract typical tetanus and quickly succumb.

The toxin, however, finally disappears from the blood though a certain portion of it may still be found in the pericardial cells and especially in the fat-bodies.

Never, under any circumstances, was I able to observe that the blood of the larvae of Oryctes exerted any antitoxic action. At the stage when this fluid no longer gives tetanus to mice, it is absolutely incapable of preventing intoxication when mixed, before injection, with tetanus toxin.

Amongst adult insects the cricket is best adapted for researches on tetanus. The field cricket will bear a temperature even higher than 30° C. It is completely resistant to injections of tetanus toxin, but it showed no more capacity than did the larvae of Oryctes or the Arachnida of producing any tetanus antitoxin.

[Sidenote: [346]]

All the Invertebrata that I have been able to study have exhibited a remarkable resistance against the known bacterial toxins, but the mechanism of this natural immunity could not be exactly made out owing to the difficulty met with in investigating the toxins in the organs and following their modifications. The idea of making use of these lower animals for the purpose of solving the problem of the origin of antitoxins is not realisable, from the fact that the Invertebrata that have been studied have never, in my experience, produced any of these substances as the result of injections, whether single or repeated, of toxins.

The natural immunity of the Invertebrata against bacterial toxins cannot therefore be regarded as an example of humoral immunity. It must be placed in the category of histogenic immunity, although we are not in a position to define accurately the part played by the cellular elements in the defence of the animal against these poisons. We must, therefore, go higher up in the animal scale if we are to solve the principal questions in regard to antitoxic immunity.

[Sidenote: [347]]

The lowest Vertebrata, the fishes, are not well-suited for this kind of research. The best known bacterial toxins act specially on warm-blooded animals and require the co-operation of high temperatures. Fishes do not live well in captivity except at relatively low temperatures and soon die if placed in an incubator kept at 30° C. or higher. It is necessary, therefore, to have recourse to the Amphibia, which are much more easily acclimatised to these temperatures. The Axolotl, coming from Mexico, is naturally capable of withstanding great heat. These animals will live for long at a temperature of 30°–37° C. They possess the drawback, however, of being very susceptible to the tetanus toxin, very small doses of it being fatal. The green frog (Rana esculenta) is the most suitable for our purpose. It readily adapts itself to optimum temperatures (30°–36° C.) and exhibits at least a certain degree of immunity against various bacterial toxins. We have stated in a preceding chapter that the green frog is unaffected by considerable quantities of diphtheria toxin. It is resistant also to tetanus toxin, but this natural immunity appears to be connected with special conditions. Courmont and Doyon[502] were the first to draw attention to the fact that beyond 20°–25° C. green frogs may contract tetanus. Refractory in winter they become susceptible in summer. These observers afterwards found that of frogs inoculated with the same dose of toxin and divided into two sets, one set kept at a temperature of about 10° C. remained quite well whilst the other set subjected to one of 30°–39° C. contracted tetanus after five days’ incubation. This experiment has been confirmed by several observers, and indicates that the tetanus poison demands, for the manifestation of its toxic action, a favourable and fairly high temperature. This result must, however, be accepted with some reserve. Undoubtedly the doses of tetanus toxin which induce fatal tetanus in frogs kept at a high temperature are innocuous when these animals are living at low temperatures. But we can, by increasing the dose, produce tetanus in frogs even when the temperature is not very high. Thus Marie[503] was able, during the whole of the winter, to tetanise both green and brown frogs living in water the temperature of which oscillated between 13° and 18° C. The incubation period in this case is very much longer (sometimes extending to 25 days) than in frogs kept at higher temperatures.

Temperature, therefore, is an important factor in the poisoning by the tetanus toxin and in the resistance of the frog, but, in the long run, this poison can exert its specific action even at relatively low temperatures.

[Sidenote: [348]]

Morgenroth[504] endeavoured to analyse the mechanism of this resistance and of the susceptibility of the green frog when maintained at various temperatures. He demonstrated that the tetanus toxin is fixed in the central nervous system, even at low temperatures, near 8° C.; under these conditions, however, it is incapable of causing the slightest tetanic symptom. When placed in an incubator kept at 32° C. the frogs contract tetanus after a period of incubation of some (2 to 3) days. During the first 24 hours of this period the frogs manifest no sign of tetanus, and if they are again put in a cool place they continue in good health. If, however, after a not too prolonged stay in the cold, these animals are subjected a second time to the higher temperature, they become tetanic, after a shortened incubation period. Cold, therefore, may arrest tetanus even at a stage when the toxin has already produced certain latent but permanent modifications of the nervous system.

Frogs injected with tetanus toxin and kept in a cold place finally get rid of the poison. When transferred to a warm chamber after a certain lapse of time they no longer contract tetanus. We have found that the greater part of the tetanus toxin continues for some time in the blood of frogs injected and kept at a low temperature. A small quantity of this blood withdrawn almost two months after the last injection produced fatal tetanus in a mouse. We do not know how frogs eliminate the toxin, but it has been demonstrated that in this case it causes no production of antitoxin. Morgenroth has confirmed this result.

[Sidenote: [349]]

Reptiles must be regarded as vertebrates exhibiting a most pronounced natural immunity against tetanus. They show an unlimited resistance to enormous doses of tetanus poison, and this at low, medium, or high temperatures (30°–37° C.). Green lizards withstand considerable doses of tetanus toxin. Although they do not contract tetanus, they get rid of the poison exceedingly slowly. Thus, a lizard kept at a temperature of 20° C., and injected with an amount of toxin sufficient to kill 500 mice, at the end of two months still retains in its blood such an amount of the poison that one-tenth of a c.c. will cause fatal tetanus in a mouse. Turtles present an analogous case. The marsh turtle, Emys orbicularis, tolerates very large amounts of tetanus toxin, injected subcutaneously, and this at both low and high temperatures, at 30° C. and beyond (36°–37° C.). The toxin passes quickly into the blood and remains localised there for a very long time. In a turtle kept in an aquarium at the laboratory the blood was tetanigenic for the mouse even four months after an intraperitoneal injection of the toxin. In another turtle which lived at incubator temperature (36°–37° C.), the blood was still toxic two months after a subcutaneous injection of tetanus toxin in quantity fatal for 500 mice. In turtles kept at 36° C. I observed abundant transudations into the peritoneal cavity, and the fluid, very poor in formed elements, was found to be very tetanigenic. It must be accepted, therefore, that the toxin is retained in the blood plasma with which it passes into the transudation. Every kind of cell must exhibit a very marked negative chemiotaxis against tetanus toxin for this poison to be retained so long in the body fluids. Under these conditions it is not surprising that in turtles I was never able to observe the slightest antitoxic power in the blood. Their great natural immunity must be due to some other factor.

The alligator (Alligator mississipiensis) has also been found to be quite refractory to tetanus both at low and at high temperatures. Outwardly alligators behave exactly as do turtles, that is to say, after the injection of various and sometimes very large doses of toxin they exhibit no morbid symptom either general or tetanic. But the particular changes which occur in their organism differ essentially from those met with in the turtle. The toxin is rapidly eliminated from the blood of the alligator, even when it is kept at a relatively low temperature (20° C.). Under these conditions of temperature, however, the blood does not become antitoxic although it has lost its tetanigenic property. When, however, the alligators are kept at a higher temperature (32°–37° C.), antitoxic power is developed in their blood, often with very great rapidity. Quite young alligators (weighing about 500 grammes) are capable of producing antitoxin, though somewhat slowly. A month after the first injection of the tetanus toxin their blood is incapable of causing tetanus in mice, but is not yet antitoxic. A month later, however, it never fails to prevent an attack of tetanus when mixed with fatal doses of the toxin and injected into mice.

Older alligators develop antitoxic power much more rapidly, and on several occasions we have found, to our great astonishment, that, as early as 24 hours after injection of the toxin, their blood was distinctly antitetanic. The blood of the same alligators, tested before the injection of the toxin, like the blood of normal alligators generally, exhibited no antitoxic property.

In several experiments we took the rectal temperature of our animals and were never able to observe the slightest rise corresponding to the temperature of the water in which the alligators lived.

[Sidenote: [350]]

It cannot be doubted then, that, in spite of the facility with which these reptiles produce tetanus antitoxin, their immunity does not depend on this antitoxic property. Thus, young alligators which have resisted a single dose of toxin sufficient to kill 6000 mice must owe their immunity to some other cause than the antitoxic power of the body fluids, for their blood does not begin to exhibit this property until two months after injection.

These same reptiles are also very refractory against cholera toxin, even in large doses; they react to the injection by the development of the corresponding antitoxin. On the other hand they are very susceptible to diphtheria toxin, small quantities of which are quite sufficient to bring about a fatal intoxication.

Snakes, like other reptiles, are refractory against tetanus toxin. In the study of their natural immunity, however, we are confronted by the difficulty that their blood is naturally toxic for laboratory animals. This toxin, analogous to the ichthyotoxin of eel’s serum, has been compared with snake venom against which the snakes themselves enjoy a very marked immunity.

[Sidenote: [351]]

Not venomous snakes only exhibit immunity against their own poison. Long ago Fontana[505] observed that non-venomous snakes resist the bite of the viper and even subcutaneous inoculation of its venom. Phisalix and Bertrand[506] confirmed these observations and were able to show that a non-venomous snake (Tropidonotus) will withstand a dose of venom capable of killing from 15 to 20 guinea-pigs. Seeking for the cause of this natural immunity, these observers came to the conclusion that it is due to the presence in the blood of toxic substances analogous to those of the venom of the viper. These same substances are found also in the labial glands of the upper jaw of the Tropidonotus and can from thence, according to the view of Phisalix and Bertrand, pass into the blood as an internal secretion. Calmette[507] has shown that the blood of snakes, injected in a non-toxic dose, vaccinates certain mammals against snake venom, and Phisalix and Bertrand have even obtained an antitoxic effect by injecting a mixture of snake’s blood, heated to 58° C., with lethal doses of venom. There is, then, in this example something analogous to what we have described in scorpions, with this difference, however, that the blood of these Arachnids is already antitoxic, to a certain degree, whilst that of snakes only becomes so after it has been modified by heat.

The classic example of immunity against a bacterial toxin amongst Birds is that of the fowl, which is highly refractory against the tetanus toxin. In the very earliest researches on this poison injections were made into vertebrates of very different kinds, and a very striking feature was the facility with which fowls resist very large quantities of tetanus toxin. However, as is almost always the case, this immunity has been found not to be absolute. By means of enormous doses, injected subcutaneously or into the muscular tissue, tetanus of the most typical kind, ending in death, has been induced in fowls, and in fowls weakened by cold, tetanic intoxication, even with smaller doses, has been set up. By injecting the toxin directly into the brain, according to Roux and Borrel’s method, the fowl may be still more easily tetanised. Thus, von Behring[508] observed that by injecting one milligramme of the toxin into the brain of a fowl, weighing one kilo, tetanus may infallibly be produced.

After the brilliant and fruitful discovery of the antitoxic property of the blood, made by von Behring in collaboration with Kitasato, we were justified in concluding that immunity against toxins and, amongst others, natural immunity, might depend on the power of the body fluids to neutralise the toxins. This hypothesis has been formulated at various times, but it was for the first time subjected to experimental control by Vaillard[509], and specially in connection with tetanus in the fowl. The blood or blood serum of these birds, when mixed in varying doses, small, medium, and large, with tetanus toxin, was never found to be capable of preventing susceptible animals (mice, guinea-pigs, rabbits) from contracting tetanus: these animals so treated behaved just as did the controls inoculated with toxin only.

[Sidenote: [352]]

The great resistance of the fowl against tetanus,—one of the most typical examples of natural immunity against a microbial poison,—cannot, therefore, be explained by the presence in the body fluids of an antitoxin capable of neutralising and rendering innocuous the tetanus toxin. On the other hand, we are not justified in attributing it simply to the absence of corresponding receptors in the sensitive nerve cells. Since the fowl readily contracts tetanus when the toxin is injected directly into the brain or when the fowl is weakened by cold, it is evident that the sensitive elements never fail to absorb and fix any poison that is presented to them. In ordinary cases, however, when the fowl exhibits its remarkable resisting power against the toxin injected in very large quantity, subcutaneously, into the muscles or into the peritoneal cavity, the poison does not reach the sensitive cells, being arrested and rendered innocuous whilst circulating in the tissues of the organism.

Von Behring[510] is of opinion that in examples of natural immunity, such as the one just examined, the principal cause of the refractory condition depends upon the impermeability to the toxin of the capillary wall of the vessels. It is, however, difficult to maintain this thesis in regard to tetanus in the fowl, when it is remembered how readily tetanus toxin passes through filters and membranes, and especially in view of the fact that weakening of the fowl by means of cold renders it susceptible to doses of toxin which are tolerated without inconvenience by normal fowls.

We are, therefore, compelled to place the natural immunity of the fowl against tetanus toxin in the category of cell immunities. This toxin, as we have said, must be arrested en route before it reaches the cells of the nerve centres. But where and how does this beneficent arrest take place? Ten years ago Vaillard demonstrated that the blood of fowls that have received an injection of tetanus toxin causes typical tetanus in susceptible animals. This tetanigenic property of the blood persists for a certain number of days. When it is measured by the quantitative method, it is found that all or almost all the tetanus toxin injected into the peritoneal cavity of the fowl passes into the blood and remains there intact for a variable number of days. From a morphological point of view the blood, immediately after the injection of the toxin, exhibits a hyperleucocytosis of greater or less duration.

[Sidenote: [353]]

When the fowls are killed at the stage when their blood becomes tetanigenic (as the result of the injection of the toxin into the peritoneal cavity), it can be demonstrated that their viscera are not capable of producing tetanus in susceptible animals except in so far as they contain blood. It is only the vascular organs, rich in blood, such as the spleen, liver, kidneys, thyroid gland and bone-marrow, that impart tetanus and then only in so far as they have not been freed from blood. Of the various organs only the genital glands, ovaries or testes, absorb a certain amount of the injected toxin. Very young testes or the smallest ovarian ova containing as yet no trace of yellow yolk, when injected into mice, produce a fatal tetanus.

In fowls, insusceptible to tetanus toxin, this toxin is found, then, in the sexual glands and in the blood. When, in order to ascertain the exact localisation of this toxin, we measure the tetanigenic power of the whole blood as compared with that of the aseptic exudations induced by the injection of gluten-casein, and necessarily much richer in leucocytes, we get the result that the exudations contain more tetanus toxin than does the blood. We are led, therefore, to the conclusion that this poison is absorbed, at least in part, by the leucocytes, and it is in these elements and in the genital cells that we must look for the factors which arrest the toxin and prevent its reaching the nerve centres.

Cellular or histogenic immunity is often contrasted with chemical immunity without taking into consideration the real analogies and differences to be found between them. It is evident that in both groups the organism of the animal modifies the introduced toxins and that this modification is a chemical process. In cellular immunity, however, this act is preceded by certain biological phenomena, such as the reaction of the formed elements and the absorption of the noxious substance. Immunity in these cases is more complex than in the example where the toxin is neutralised by a direct action of the body fluids, but ultimately it always resolves itself into a chemical or perhaps physico-chemical action of the substances of the organism of the animal on the toxic substances of the poisons.

[Sidenote: [354]]

In Mammals examples of natural immunity against certain poisons are not rare. Almost a century ago Oken made the observation that a person who tried to poison a hedgehog with opium, hydrocyanic acid, arsenic or mercury bichloride usually failed in his attempts because of the great resisting power of this animal. Harnack demonstrated that the hedgehog will withstand a dose of potassium cyanide six times as great as that necessary to kill a cat in a few minutes (0·01 grm.). In Lewin’s[511] experiments the hedgehog was found to resist the injection of powdered cantharides in a quantity seven times as great as that which infallibly kills a dog and greater also than the lethal dose for man. The same observer also confirms the observation that a much larger dose of alcohol must be used in order to intoxicate a hedgehog than is required to obtain the same effect in the rabbit or even in the dog. Horvath[512] fed hedgehogs for a fairly long period with living cantharides. These Insectivora devour their venomous prey without showing any sign of illness except a certain degree of emaciation. When Lewin tried to ascertain the cause of this natural immunity of the hedgehog he examined the blood of this animal for a substance antitoxic to cantharidine. His experiments were all negative; but it is difficult to come to any definite conclusion in this matter from the fact that the blood and blood serum of the normal hedgehog are toxic for the small laboratory animals. A similar objection had already been brought forward by Phisalix and Bertrand in connection with their experiments, analogous to those of Lewin, on the immunity of the hedgehog against the venom of the viper.

[Sidenote: [355]]

It has long been known that hedgehogs have a liking for certain reptiles and wage an implacable war on snakes in general and on the viper in particular. In its attack the hedgehog tries to avoid being bitten, but when, as often happens, it fails to evade a bite the inoculation of the viper’s venom appears to be well borne. This observation has been confirmed experimentally. Phisalix and Bertrand[513] have shown that the resistance of the hedgehog to the viper’s venom is about forty times as great as that of the guinea-pig, that is to say the hedgehog, though far from possessing an absolute immunity, nevertheless exhibits a much greater resistance than do most animals. Lewin[514] convinced himself of this fact as regards adult hedgehogs, though young animals, according to him, are much more susceptible. Thus, he has seen a young hedgehog that had been bitten by a viper die after nine days’ illness. This observation speaks in favour of the conclusion that the immunity of the hedgehog might be naturally acquired rather than a really natural immunity. The hedgehog, hunting all kinds of small animals, might often be bitten by vipers and in this way acquire its immunity against the venom. Under these conditions we can readily conceive that the blood of this “insectivoran” might be placed in a position to develop a specific antitoxic property.

When Lewin tried to satisfy himself of the existence of this property by direct experiment he could only show that the blood of the hedgehog was powerless to prevent the lethal effect of the viper’s venom on small animals. But here, as in his researches on cantharidine, he did not take into account the inherent toxicity of the blood of the hedgehog. Phisalix and Bertrand[515], who have also studied this question, have obtained results at variance with those of Lewin. They demonstrated first of all that the blood of normal hedgehogs was capable of intoxicating and even of killing laboratory animals such as the guinea-pig. It is quite natural, therefore, that the mixture of this fluid with viper’s venom could not be tolerated. It was, however, sufficient to heat the blood of the hedgehog to 58° C. for it to become not only innocuous of itself, but even for it to exhibit an antitoxic action against snake venom. Thus, guinea-pigs which had received 8 c.c. of heated hedgehog’s serum into the peritoneal cavity, were at once in a condition to resist double the lethal dose of viper’s venom. Phisalix and Bertrand conclude, therefore, that “the natural immunity of the hedgehog against the viper’s venom is due to the presence in its blood of an immunising substance.” The same observers[516] satisfied themselves that horse’s serum and even that of the guinea-pig exercise an undoubted antivenomous action; yet these animals are anything but insusceptible to snake venom. Moreover, the necessity to heat the blood to 58° C., as a preliminary measure, deprives this conclusion of the degree of certainty one would like to have in such a matter. On the other hand, the greater susceptibility of young hedgehogs prevents us from putting the immunity of the adult in the category of natural immunity properly so called.

[Sidenote: [356]]

Analogous considerations apply in the case of the mongoose (Herpestes ichneumon), carefully studied by Calmette[517], according to whose researches the Antilles mongoose is not very susceptible to snake venom; it readily withstands doses very large relatively to its size, but its immunity is not absolute. It owes much of its mastery in its fights with venomous snakes to its extraordinary agility. The blood of the mongoose, mixed with venom, exhibits an undoubted antitoxic power, though this is not sufficient to prevent the death of susceptible animals. We have no data to enable us to explain the origin of this antitoxic property, but it is probable that here again we have an example of relative immunity, acquired during life. Calmette points out, however, that his ichneumons came from Guadeloupe, where no venomous snakes are found. We may, of course, suppose that the feebly antitoxic power of the blood of these mammals might be due to other snakes or to species of animals whose blood possesses a certain venomous property[518].

[Sidenote: [357]]

We have far more exact data on the natural immunity of certain mammals against toxins of microbial origin. The example most thoroughly studied, one which has become, one might say, classic, is that of the rat against diphtheria toxin. Since the discovery of this toxin, the first well-studied bacterial poison, a discovery made by Roux in collaboration with Yersin, it has been recognised that mice and rats tolerate large quantities of diphtheria cultures or of their filtered products. A rat resists a dose of the diphtheria poison capable of killing several rabbits. To explain this great natural immunity it was suggested that the antitoxic property of the body fluids could be called in. It was supposed that the rat’s blood was, by its very nature, endowed with the power of neutralising the toxin of diphtheria. But, as in the tetanus of fowls, it was not long before facts rendered this hypothesis untenable. Kuprianow[519] studied this question under the direction of Loeffler and gave an account of the results of his experiments, which proved that the blood of the sewer rat, which is very refractory against diphtheria, contains no substance that will neutralise the morbific action of diphtheria toxin on susceptible animals, especially the guinea-pig.

It was necessary to seek some other explanation, and the idea that the immunity of the rat depends on the insusceptibility of its living cells to the diphtheria poison was seized upon. The experiments carried out by Roux and Borrel[520] demonstrated the incorrectness of this hypothesis. The immunity of rats to subcutaneous or intraperitoneal injection of diphtheria toxin is very marked. But a very small dose (0·1 c.c.) of this poison, introduced directly into the cerebral substance of the rat, produces a complete paralysis, which lasts for several days, and ends in the death of the animal. Roux and Borrel conclude from this “that the brain of the rat is specially sensitive to the action of the diphtheria poison, and that as this animal does not die as the result of the injection of large quantities of toxin into the subcutaneous tissue, it is because the toxin does not reach the brain.” These authors have pointed out analogous facts in connection with other examples of natural immunity. The rabbit, which withstands a hypodermic injection of 30 centigrammes of chlorhydrate of morphia, is killed by 1 milligramme only of this salt, introduced directly into the brain. Here, again, neither the cellular insusceptibility nor the antitoxic property of the blood (no “antialkaloidal” power could ever be demonstrated) can explain the immunity, which appears to be due rather to the factor which arrests the poison on its way to the nerve centres.

[Sidenote: [358]]

In spite of the insufficiency of our knowledge as regards natural immunity against soluble poisons we are quite justified in affirming that this category of phenomena comes mainly into the domain of the cells. The body fluids of animals which exhibit this immunity have been found to be antitoxic in a few species only (scorpion, snake, hedgehog, mongoose). And for the majority of these it is possible to invoke special causes, such as the internal secretion of snake and scorpion venoms by the glands which manufacture them, or the acquisition of an antitoxic power during life resulting from wounds or from the absorption of venomous food. The theory of the insusceptibility of the cells of animals naturally refractory to toxins must also be rejected; it is incompatible with well-established facts. Nothing remains, then, but to assume that the formed elements are the principal factors in this natural immunity, and that they interpose to prevent the passage of the poisons towards the very susceptible nerve cells.

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