This discovery was soon confirmed by R. Pfeiffer[403] for the cholera vibrio. Animals vaccinated against this organism furnished Pfeiffer with a serum which, whilst not at all antitoxic, was distinctly protective when injected into normal guinea-pigs. It protected these animals from a fatal infection by the vibrio and, when injected into the peritoneal cavity, it set up the granular transformation of the cholera vibrios,—Pfeiffer’s phenomenon. Pfeiffer, for this reason, gave to the protective antivibrio serum the name of bactericidal serum. As the granular transformation was produced, under the influence of this serum, with cholera vibrios only and never with other species of vibrio, Pfeiffer gave to the active substance in the serum the name of specific cholera antibody. This substance, according to his theory, was formed in the animal body at the expense of an inactive antibody which became transformed into an active substance under the influence of the peritoneal endothelium.
The possibility of thus vaccinating susceptible animals by means of the serums of immunised animals, quite apart from any antitoxic power, was easily confirmed and extended to several other infective diseases. Pfeiffer and Kolle[404], Funck[405], Chantemesse and Widal[406] demonstrated it in connection with the experimental disease produced in animals by the typhoid bacillus; Loeffler and Abel[407] for the Bacillus coli, etc. The protective or anti-infective power of the serum and other fluids of immunised animals was soon recognised as a general property.
Pfeiffer and his collaborators, as well as many other investigators, laid special stress on the bactericidal character of these protective fluids. It was seen that the serums of immunised animals were often almost or completely incapable of killing the corresponding micro-organisms, but they were still regarded as bactericidal, because, when injected into the peritoneal cavity of normal animals, they set up the transformation of vibrios into granules, or, in the case of other bacteria, determined certain phenomena of extracellular destruction. Whilst carrying on researches in this direction, Fränkel and Sobernheim[408] discovered a fact of great importance. They found that the protective substance of the serum of animals vaccinated against the vibrios resisted heating to 70° C. When submitted to the influence of this temperature, the serum lost its bactericidal power completely, but remained quite as protective as the unheated serum, when injected into susceptible animals. This experiment, which has since been confirmed repeatedly, furnished us with a means of separating the bactericidal power from the protective power in cases where both were present in the same serum. Later, in the hands of Bordet, it proved to be of great service in connection with his researches on the concurrence of two substances in acquired immunity.
The possibility of obtaining Pfeiffer’s phenomenon outside the body by “reactivating” the protective serum with peritoneal fluid or blood serum of normal unvaccinated animals has still further facilitated the study of the action of the two substances in acquired immunity. It was with the help of this method that Bordet was able to furnish so much valuable information on the subject of anticholera serums and, later, on that of haemolytic serums. The discovery by Ehrlich and Morgenroth[409] of the fixation by the sensitive elements of the heat-resisting (thermostabile) substance (that which resists a temperature of 65°–70° C.) constitutes a new and important acquisition to the study of acquired immunity. The discovery has been applied by Bordet to micro-organisms, and since then it has been found possible to study much more precisely the mode of action of specific protective serums.
Even before this last scientific advance had been made it was possible to determine the relations between the protective power and the agglutinative power of the fluids of animals that had acquired immunity. Both resist about the same temperatures; both are found in the blood plasma and pass into the fluids of exudations and transudations. But it may be affirmed with certainty, as already stated, that the two properties are quite distinct. Pfeiffer has laid great stress on the fact that highly protective serums often exhibit only a feeble agglutinative power and vice versa. During an investigation[410] into an epidemic of typhoid fever, he had occasion to study the serum of patients convalescent from this disease. The exact dosage of the two properties demonstrated that a slightly marked agglutinative property might be associated with a very powerful protective property. Gheorghiewsky[411] made similar observations on animals vaccinated against the Bacillus pyocyaneus. The serum of a goat, although more agglutinative, invariably proved to be less protective than that of a rabbit. A similar result was obtained with the serum of immunised guinea-pigs. “This shows distinctly”—concludes Gheorghiewsky—“that the property possessed by serums of agglutinating the Bacillus pyocyaneus does not march parallel with the protective property” (p. 304). Analogous examples are sufficiently numerous to justify us in accepting the distinctiveness of the two properties of specific serums.
The protective or anti-infective substance is, therefore, not the same as the agglutinin. But are we justified in regarding it as identical with the fixative substance, or fixative (sensibilising substance, immunising or intermediary substance, or amboceptor)? From the fact that the fixative was at first rightly designated by Bordet as protective substance we should conclude in the affirmative. The question is an important one and merits close examination. The discovery of an exact method of determining the presence of fixatives has rendered it possible to ascertain whether these substances are always found in the protective fluids and also whether the presence of fixatives necessarily implies the protective power of the serums.
The first of these questions has been answered in the affirmative. All the protective serums studied from this point of view, by Bordet and Gengou, were found to be endowed with very distinct fixative properties. They also found the specific fixative in the serum of guinea-pigs immunised with the attenuated bacilli of the first vaccine of Pasteur. Now this serum is powerless to prevent the production of fatal infection in mice into which is simultaneously injected the bacillus of the first vaccine. Consequently a fixative fluid is not necessarily protective. This is in accordance with the fact that the micro-organisms that have absorbed the fixative may, nevertheless, retain their virulence. We have already cited the experiment of Mesnil that the bacilli of swine erysipelas, mixed with the specific serum and then deprived of this fluid, produce a fatal infection in mice. We have also drawn attention to the fact, demonstrated by Sawtchenko, that anthrax bacilli, obtained from the exudation of immunised rats, give rise to a fatal anthrax in normal guinea-pigs and rats. The experiments of Bordet and Gengou proved that there is absorption of the fixative substance by the bacilli of swine erysipelas and of anthrax when placed in contact with the specific serums of the immunised animals. In order that the protective power may manifest itself adequately, therefore, besides the fixative substance, some other factor capable of acting is also necessary.
In connection with my work on immunity against the micro-organism of swine pneumo-enteritis I was able to demonstrate that the serum of vaccinated rabbits, incapable of preventing the multiplication of the specific cocco-bacillus, is also powerless to deprive it of its virulence; it is without the power of causing its agglutination or of neutralising its toxin. In short, this serum appears to exercise no direct action on the micro-organism, yet, in spite of that, it prevents its pathogenic action. With these results before me, I was led to assume a certain stimulating action of the serum on the defensive elements of the animal organism and especially on the phagocytic system. The discovery of the fixative property of serums would lead us to believe that this stimulation was entirely useless, and that the permeation of micro-organisms by the fixative was amply sufficient to bring about their destruction and removal from the animal. A living micro-organism in its normal form, endowed with full virulence and provided with its fighting weapon, the toxin, but at the same time permeated by the fixative substance, might behave in the animal in some special way. It might excite a strong positive chemiotaxis of the leucocytes and be ingested and destroyed by these cells with greater facility. A priori, there would be nothing to object to in this view, but certain facts are opposed to it. Thus, in the case of micro-organisms just cited, we see bacteria, permeated not only with the fixative but also with cytases, capable of producing a fatal infection. We are thus compelled to accept the theory of an influence of protective serums not only on the micro-organisms but also on the organism of the animal into which they are introduced. As this influence manifests itself in the form of a strong phagocytosis, it is only natural that we should attribute it to the existence of a stimulating action of the serums of vaccinated animals on the phagocytes of the normal animals. The detailed analysis of the mechanism of the immunity acquired as the result of the injection of these serums, as we shall attempt to prove in the following chapter, in many cases confirms this view.
The important part played by the stimulation of the phagocytic reaction in acquired immunity is supported by yet another series of facts and from a different side. It has been clearly established that not only the serum of immunised animals but also that of normal man and normal animals, themselves susceptible to the pathogenic action of the micro-organisms, protects the animal organism against infection. This fact was first demonstrated in connection with researches on the vaccination of guinea-pigs against the experimental peritonitis produced by the cholera vibrio.
G. Klemperer[412] was the first to observe that the blood of several individuals who had never had cholera was, nevertheless, in the case of guinea-pigs, protective against peritoneal infection by the cholera vibrio. He concluded therefrom that the individuals who had furnished this protective blood possessed immunity against cholera. Soon afterwards I[413] was able to extend analogous researches over a large number of persons and to show that the protective power of the blood is of very wide distribution in human beings. But, instead of assuming that all these individuals, whose fluids protect the guinea-pig from peritoneal infection, possess a natural immunity against cholera, I came to the conclusion that the protective power of the blood cannot be taken as a measure of the immunity of the individual from whom the blood was drawn. Here again I assumed a stimulant action of the human blood on the phagocytic reaction of the guinea-pig, looking upon it as quite natural that the blood, capable of exciting the reaction in an alien animal, might remain inactive in the body of the animal which furnished it.
R. Pfeiffer[414] has given much attention to the protective action of serums; he has laid special stress on the essential difference between the influence of normal serums and of those obtained from animals that have acquired immunity. Whilst, in order to obtain a protective effect with the normal blood or serum of man and animals, it is necessary to inject a considerable quantity (from 0·5 c.c. upwards), the specific serum, i.e. serum obtained from persons recovered from cholera or from animals vaccinated against the cholera vibrio, is active in a very minute dose. Sometimes the cholera peritonitis of the guinea-pig is prevented by a fraction of a milligramme of such serum[415]. Based on these facts, Pfeiffer has expressed the view that the normal serum acts by stimulating the natural powers of defence of the animal, whilst the specific serum exercises its influence in virtue of the property of causing the formation of a special secretion which acts only against the micro-organism which served for the production of the immunity. Pfeiffer and his collaborators have demonstrated that normal serums are protective, not only against the cholera vibrio, but also against several other micro-organisms, e.g. the typhoid bacillus. One of his pupils, Voges[416], believed that, in certain infections, the protective power of normal blood might be greatly exaggerated, and that, in these cases, the limit between the activity of normal and of specific serums might be almost completely effaced. He affirmed, especially, that very small doses (0·1 c.c.) of blood serum from a normal guinea-pig was quite sufficient to prevent, in other guinea-pigs, a fatal infection by the micro-organism of hog cholera and its allies. As this fact might be of general application I asked M. Saltykoff[417], who was working in my laboratory, to verify the statements of Voges. Several series of experiments demonstrated the incorrectness of the contention. The small doses of normal serum of guinea-pigs, indicated by Voges, were found to be absolutely incapable of protecting against the virus used by him in his experiments.
The fact that normal serums, injected in sufficiently large doses, exhibited an undoubted protective property, affords additional proof that this property cannot be identified with the fixative power. The latter was present in serums which were not protective; here, then, we have the inverse phenomenon and we see normal serums exercise their protective action although they contain no fixative. This follows from Bordet and Gengou’s experiments already described, according to which the cytases, placed in contact with micro-organisms in normal serums, remain free, simply because of the absence of fixatives.
We are led, then, from these demonstrations to recognise the presence of stimulins not only in specific serums, but also in normal serums. Between the two there is this difference that, when applied with the normal fluids, the stimulins alone act, whilst when injected with the serum of the animal enjoying acquired immunity the action of the stimulins is facilitated and reinforced by the fixatives or sometimes, perhaps, by the agglutinins.
The stimulating influence of certain normal serums may be so considerable that it may prevent infection by the micro-organism, injected at the same time in a dose many times more than lethal. Wassermann[418] protected guinea-pigs by injecting into the peritoneal cavity a quantity as great as 40 times the lethal dose of typhoid bacilli, by introducing at the same time and at the same place 3 c.c. of normal rabbit’s serum, heated to 60° C. Besredka[419], who confirmed this observation, has analysed its special mechanism. He showed that the serum exercises a very marked stimulating influence on the guinea-pig’s leucocytes, which then exhibit a truly extraordinary phagocytic activity. They are seen to act in the peritoneal fluid, but they are much more active in the region of the omentum, where the leucocytes gorge themselves with micro-organisms, devouring them by dozens. The stimulating action of the heated rabbit’s serum is exercised in a similar fashion if, instead of micro-organisms, grains of carmine be injected. Very shortly after the commencement of the experiment very little carmine is found outside the cells; it is all either ingested by individual leucocytes, if the grains are small, or surrounded by numerous leucocytes when the grains are massed together; this phagocytosis is most developed in the region of the omentum, exactly as in the case of typhoid bacilli.
These facts, which so clearly demonstrate the stimulating action of the normal rabbit’s serum, prove in another way that the stimulin resists heating to 60° C., and that, in this respect, it resembles the agglutinins and fixatives. This may afford us an indication as to the nature of the stimulating substance. The possibility of obtaining an antistimulin gives us another valuable indication. Wassermann, in the work we have just cited, showed that the serum of a rabbit, previously treated with guinea-pig’s serum and injected under the same conditions as in the experiment with normal rabbit’s serum, has completely lost its protective power. The typhoid bacilli multiply freely in the peritoneal cavity and the organism of the guinea-pig is incapable of opposing a sufficient resistance. Wassermann thinks that, in this case, the disease becomes grave because of the anticytase found in the serum of rabbits treated with guinea-pig’s blood. There is no doubt that this serum is really anticytasic. But as the free cytases found in the peritoneal cavity of a guinea-pig inoculated at the moment of phagolysis, become inactive under the influence of the anticytase and play merely a minor part, it is impossible to accept the German investigator’s interpretation. Indeed, Besredka has proved that, in this case, it is the antiphagocytic or antistimulant action of the rabbit’s serum which brings about the fatal issue in the case of the typhoid inoculation.
We have laid stress on the point that an animal, whose serum is protective when introduced into another animal, may itself not be refractory against the specific micro-organism. As regards the serum of normal unvaccinated animals this has been so fully demonstrated that nowadays no one doubts it. The question is more complicated in the case of animals that have acquired immunity. As in the great majority of cases the serum of these animals is found to be endowed with a very great protective power, it has been accepted as proved that the animal which furnishes it must itself possess great immunity. The degree of protective power has even been taken as the measure of the acquired immunity. Thus, the numerous attempts to vaccinate the human subject against typhoid fever, undertaken in consequence of the researches of Pfeiffer and Kolle[420], were based on the fact that in these cases the serum of vaccinated individuals acquires a great protective power. It was argued that if this power is present it can only be due to the acquired immunity of the individuals who furnish such a serum. Undoubtedly the protective property of the fluids and the resistance are often equal; but it is none the less true that there are cases where, in spite of this property being markedly developed, the animal that furnishes the protective serum is susceptible to the action of the micro-organism and may even succumb to infection therewith.
As the hypothesis just mentioned is of importance from a general point of view it must be supported by adequate proof. It was during the course of the vaccination of rabbits against the micro-organism of the pneumo-enteritis epidemic at Gentilly that I was first able[421] to assure myself of its accuracy. I noticed that some of these rabbits, although vaccinated, ultimately succumbed to pyaemia, set up solely by this micro-organism. They were consequently not refractory against the disease, and yet their blood serum, when injected into normal rabbits along with an absolutely fatal dose of micro-organisms, was found to be highly protective. This observation drove me to the conclusion that the protective power is not a function of immunity and cannot be received as a measure of this immunity. Analogous facts have since been demonstrated in certain other cases. Thus, Pfeiffer[422] on several occasions has found that guinea-pigs, highly immunised against the cholera vibrio, have succumbed after the injection of a moderate quantity of these organisms. “On post-mortem examination of these cases living vibrios were found in the peritoneal cavity, sometimes in considerable numbers; and yet minimal doses of the heart blood given to normal guinea-pigs caused in these animals a very marked breaking down of the vibrios.” Alongside these facts may be placed others, described in the preceding chapter, of well immunised animals dying from infection, after they had been weakened by opium, cold, or other lowering agent. It is clearly seen, then, that for the manifestation of acquired immunity it is necessary that the reaction of the living cell elements should take place without let or hindrance. When this reaction fails, the possession of even great protective power is insufficient to prevent the immunised animal from contracting a fatal infection.
If, in acquired immunity against micro-organisms, it is really the cell defence which plays the most important part, we can readily imagine cases where it by itself can confer immunity without calling in the co-operation of the protective power of the fluids. When in this connection we study the resistance of an animal against various pathogenic organisms, we note, first of all, the very great variability that exists in the production of the acquired humoral properties. In certain cases, as in vaccination against vibrios or typhoid bacilli, the serum very readily becomes not only protective, but agglutinative and fixative. In other cases these properties develop with difficulty and are only manifested after a long period of vaccination. Such is the case with anthrax. After the discovery of protective serums, numerous attempts were made to obtain a serum protective against the anthrax bacillus. Several observers failed in their attempts, others were more fortunate. Sclavo[423] and Marchoux[424] were the first to succeed in obtaining a protective serum from animals hyperimmunised against anthrax. They were able to show that the serum of sheep, treated first with vaccines and then repeatedly with anthrax virus, would protect rabbits against a fatal dose of the bacillus. Marchoux even obtained, with hyperimmunised rabbits, a serum which prevented normal rabbits from contracting fatal anthrax. Sobernheim[425] was less fortunate in his first experiments. He satisfied himself that the blood serum of cattle that had recovered spontaneously from anthrax or that had been vaccinated according to Pasteur’s method, was absolutely unable to protect small animals against the anthrax bacillus, and his hypervaccinated rabbits furnished serums of doubtful activity. It was only later that he succeeded[426] in obtaining better results; especially when he used sheep. Even then he found that in the production of the anti-infective property the individuality of the immunised animals had a dominant influence. Thus, in two sheep, treated in exactly the same way, the serum of one was found to be incapable of protecting a rabbit, whilst that of the other exhibited an undoubted, although feeble, protective power.
But what is of greater interest to us, from our point of view, is that guinea-pigs which have been vaccinated against anthrax and which enjoy a considerable immunity against this disease, exhibit no protective power. In a letter from Behring I learnt that this fact had for the first time been demonstrated by Wernicke in experiments carried out in the Hygienic Institute at Marburg. After repeated and painstaking attempts this observer succeeded in vaccinating guinea-pigs against enormous doses of virulent anthrax bacilli. The serum from the animals so immunised was, however, quite incapable of protecting normal guinea-pigs against a fatal infection. This result was the more extraordinary since Wernicke’s pigeons, likewise vaccinated against anthrax, gave a serum whose protective power was quite distinct. Realising the great importance of these facts I asked M. de Nittis[427] to repeat these experiments in my laboratory. The vaccination of pigeons is an easy matter, but that of guinea-pigs presents great difficulties. He succeeded, nevertheless, in vaccinating some of these rodents very highly, and this enabled him to compare the protective power of the blood serum in the two species. That of the vaccinated pigeon was found to be endowed with this power and protected guinea-pigs and mice against virulent anthrax. The serum of the immunised guinea-pigs, on the contrary, exhibited no protective property, just as in Wernicke’s experiments. The guinea-pigs and mice, into which this serum was injected at the same time as the anthrax bacilli, died even when attenuated anthrax was used. We have, then, in this case, an example of acquired immunity, independent of any protective power of the fluids of the body.
In the course of their researches on the bacillus isolated by R. Pfeiffer from persons attacked by influenza, Delius and Kolle[428] tried to vaccinate susceptible animals (guinea-pigs) against this minute organism and to immunise animals naturally refractory (dog, sheep, goat) against fairly large doses of cultures. They succeeded in vaccinating guinea-pigs against ten times the lethal dose, but never obtained any protective serum. Nor did the other animals that were treated furnish a protective serum. “From the whole of our experiments carried on for several years”—conclude Delius and Kolle—“it is quite evident that we were unable to produce any appreciable change in the blood by the use of those methods which have produced specific immunising serums against other bacteria such as the bacilli of diphtheria, cholera, typhoid fever, and ‘blue pus’” (p. 345). Slatineano undertook a detailed study of Pfeiffer’s bacillus in my laboratory, but he found it impossible to demonstrate any unquestionable protective effect exerted by the blood serum of vaccinated guinea-pigs upon normal guinea-pigs inoculated with a fatal dose of this organism. We are not justified, therefore, in classing this bacillus with the anthrax bacillus; we may, however, cite it as an argument illustrating the difficulty that is met with, in certain examples of acquired immunity, of discovering the protective power, when feeble and masked.
The inoculation with micro-organisms of animal nature causes the development of acquired immunity, but in this case the properties of the fluids of the body are but little in evidence or they may be even nil. Let us return to the example of the Trypanosoma of the rat which excites in vaccinated animals a protective and weakly agglutinative power of the serum. This fluid, however, is usually found to be incapable even of rendering the flagellated parasites motionless.
The question of immunity against malaria has been much discussed. It is well known that a first attack of this disease, so far from conferring any immunity of the slightest durability, leaves a certain predisposition to another attack. In spite of this the study of malaria in various countries and in individuals belonging to different races has demonstrated that there does indeed exist a certain degree of acquired immunity against this disease. During recent years Koch[429] has paid special attention to this subject and has furnished us with very valuable data, based especially on a comparative study of the blood of children and adults. The frequency of Laveran’s parasite in the former and its rarity in the latter, have led him to the conclusion that infantile malaria sets up an immunity which persists in the adult. Moreover, it has been established that in malarial countries the indigenous inhabitants exhibit an attenuated form of the disease, unaccompanied by acute attacks, but with phenomena that are chronic and very slow in development.
In spite of the existence of a certain degree of acquired immunity against malaria, all attempts to demonstrate any protective action of the serum have been fruitless. Celli[430], indeed, injected, as a preventive, the blood serum of individuals who had recovered from malaria or of others who were bled during the period of defervescence following an acute crisis of this disease, but in every instance these injections were found to be useless in preventing an attack of malaria.
We can readily understand that in a disease which is exclusively human, such as malaria, it has not been possible to perform a sufficient number of experiments to decide the question of the protective property of the blood. In this respect we shall have greater chance of obtaining satisfactory data if we direct our attention to some analogous disease attacking one of the lower animals. Such a disease we have in Texas fever, occurring in the Bovidae, as the result of the action of an animal parasite, Piroplasma bigeminum, which invades the red blood corpuscles much as Laveran’s parasite invades those of the human subject.
As mentioned in the preceding chapter, Smith and Kilborne and Koch have demonstrated that the Bovidae may acquire a real immunity against Texas fever. Nicolle and Adil Bey[431] at Constantinople found indigenous races that exhibited a remarkable immunity against the Piroplasma. Having demonstrated this fact the idea occurred to them to inoculate these refractory cattle with very large quantities of virulent blood and to make use of the serum from animals so treated for the prevention of infection in susceptible races of Bovidae. This experiment gave negative results. Lignières[432] elaborated a special method of vaccinating susceptible Bovidae and was successful in obtaining very encouraging results. A commission of veterinary surgeons from Alfort[433] appointed to verify these observations came to the conclusion that “the vaccination as carried out by Lignières was absolutely effective.”
Lignières also carried out researches on the protective power of the blood serum of his immunised cattle. In a communication to the International Congress of Medicine, held in Paris in 1900, he stated that the injection of several hundred cubic centimetres of this fluid did not protect normal animals against infection. We must conclude, therefore, that, here also, we have another example of acquired immunity unaccompanied by the presence of any protective property of the blood fluid.
These results have received confirmation from a most authoritative source. Nocard has kindly communicated to me the fact that he has tried in vain to confer immunity on normal dogs into which he has injected blood serum coming from dogs that had recovered from the disease produced by a haematozoon closely allied to that of Texas fever or serum from sheep immunised with blood from the affected dogs.
Looking at the data we have just summarised as a whole, we are compelled to recognise that, on the one hand, the protective power of the body fluids may coincide with a susceptibility to the corresponding micro-organism, and that, on the other, real acquired immunity may exist without any manifestation of this humoral property, especially as, even in immunised animals, the acquired immunity often persists longer than does this property. It must be accepted then, that, in this immunity, there exists something other than the powers of the fluids of the body, that is to say, the factor which plays the predominant part is to be sought for in the cellular elements. We need only recall the many facts collected in the preceding chapter to be convinced that in acquired immunity phagocytosis is the most constant and most general phenomenon. We find it in cases where the humoral properties are the most marked, as well as in those in which they are only slightly developed or are entirely absent. We need not again discuss Pfeiffer’s phenomenon analysed in the preceding chapter. It is sufficient to mention that this example of the extracellular destruction of micro-organisms only occurs under limited and special conditions. It is observed only in cases where the injection is made into a situation rich in leucocytes which undergo phagolysis as a result of the sudden change brought about in their conditions of existence. Further, this phenomenon is observed only in connection with micro-organisms that are slightly resistant to the influence of the microcytases. In those cases in which we meet with Pfeiffer’s phenomenon, we also meet with a widely extended phagocytic reaction.
This reaction is most pronounced where the properties of the body fluids are only slightly developed or are absent. The study of acquired immunity against anthrax provides us with a very convincing proof of this. We have already cited the example of vaccinated rabbits and rats in which phagolysis is incomparably greater than in the susceptible control animals which contract a fatal anthrax. This rule is general. It is confirmed in the vaccinated sheep and guinea-pig. The absence, or feeble development, of the protective power of the blood or of the other humoral properties in no way, then, prevents the considerable change which is set up in the phagocytes of animals that have acquired immunity against anthrax. The negative chemiotaxis of the leucocytes, so marked in susceptible animals, is modified into positive chemiotaxis as the result of vaccination. This fact, one of fundamental importance, was first demonstrated for the immunity against anthrax, later being extended to other micro-organisms. Massart[434] studied the general subject and collected a series of data which led him to say that “vaccination effects an education of the leucocytes; these latter become so adapted that they can approach the virulent micro-organisms.” The best method of forming an estimate of the change which the leucocytes undergo is by injecting subcutaneously very virulent micro-organisms capable of setting up a generalised infection. The anthrax bacillus, Gamaleia’s vibrio, the streptococci and the cocco-bacilli of swine and fowl cholera are very suitable for such study. These micro-organisms, when inoculated subcutaneously into susceptible animals, set up a very slight local reaction or none at all, in the form of an exudation of transparent fluid almost entirely without leucocytes. The micro-organisms grow freely in these exudations and soon invade the animal. In vaccinated animals the local reaction is more marked and the exudation, very rich in leucocytes, is poor in fluid; the micro-organisms remain free for a very short time, being soon ingested by the leucocytes. Their destruction, inside these cells, takes a longer or shorter time according to circumstances; but in the end it is always complete.
The difference as regards phagocytic reaction between susceptible and vaccinated animals, such as I have just described, has been generally recognised by many observers. A few opponents are still found, however, who consider that they are justified in affirming that the negative chemiotaxis of the susceptible animal does not exist and that, consequently, vaccination can in no way change it into positive chemiotaxis. Werigo made himself the spokesman of this view, which he has maintained in several papers[435]. Instead, however, of introducing the virulent micro-organisms into the subcutaneous tissue of susceptible animals he injected them directly into the veins. Using cultures of the anthrax bacillus and of the cocco-bacillus of fowl cholera he injects these into the venous system of normal rabbits. The animals soon die from general infection. If, however, these animals are killed shortly after inoculation, it is found on examination of sections that many of the micro-organisms have been ingested by the leucocytes. Werigo concludes from these facts that in the higher animals the chemiotaxis is always positive; but that it ends in the destruction of the micro-organisms in the vaccinated animals, never bringing about this result in susceptible animals. Taking all the data on this question into consideration, it is easy to convince oneself that this view cannot be accepted as correct, for not only the definite phenomena observed below the skin but also the no less demonstrative process appearing in the peritoneal cavity prove most clearly the existence of this negative chemiotaxis of the leucocytes. I need only recall Bordet’s experiment on the fate of streptococci and Proteus vulgaris when injected together into the peritoneal cavity of guinea-pigs. Whilst the Proteus bacilli at the end of a very short time are all ingested by the leucocytes, the streptococci remain free in the peritoneal fluid up to the death of the animal. The leucocytes which exhibit a positive chemiotaxis as regards the former, manifest a negative chemiotaxis as regards the streptococci.
In spite of the great force of these arguments, the discovery of a means of reconciling the results obtained from the inoculation of micro-organisms subcutaneously or into the peritoneal cavity, with those observed after they had been injected into the blood vessels would be of great interest, and Zilberberg and Zeliony[436] have undertaken a series of experiments with this object. Following Werigo they made use of the cocco-bacilli of fowl cholera, and found, in accordance with his observations, that the intravenous injection of these organisms, obtained from cultures in nutrient media, causes a very marked phagocytosis of the cocco-bacilli. When, however, they injected into the veins of rabbits cocco-bacilli that had been grown in the peritoneal fluid of other rabbits, they found the micro-organisms free in the blood plasma and observed only a very restricted phagocytosis in the microphages of the liver. It follows from these experiments that the ingestion of the cocco-bacilli, in Werigo’s experiments, was dependent on the presence of a large number of attenuated micro-organisms which were present in the cultures that he employed for his injections. Alongside these organisms, slightly or not virulent, were others, endowed with their normal pathogenic activity and quite numerous enough to set up a fatal infection. When Zilberberg and Zeliony replaced cultures on agar by the peritoneal exudation which contained virulent cocco-bacilli almost exclusively, the phagocytosis in rabbits, injected into the veins, was found to be almost suppressed. With the object of establishing whether the absence of the phagocytic reaction, in this case, really depended on negative chemiotaxis on the part of the leucocytes, the above cited observers performed the following experiment. They injected into the vein of a rabbit, already affected with a generalised infection by the cocco-bacillus of fowl cholera, an innocuous culture of a saprophytic staphylococcus. Post-mortem examination showed that these cocci were almost entirely ingested by the same phagocytes which refused so energetically to seize the cocco-bacilli. This experiment, analogous to that of Bordet on streptococcus and Proteus, compels us to reject Werigo’s conclusions as to the absence of negative chemiotaxis in the phagocytes of the higher animals. I ought to add that the work of Zilberberg and Zeliony was in part executed in my laboratory so that I was able to convince myself by ocular demonstration of the complete accuracy of their statements.
Independently of these observers and even before their work appeared, Th. Tchistovitch[437] published an interesting study on the same question. He injected very virulent streptococci into the ear vein of rabbits. These micro-organisms set up a generalised and fatal infection in which phagocytosis was completely absent or nearly so. Here again was manifested a negative chemiotaxis of the phagocytes, which, henceforth, could no longer be questioned.
In certain infective diseases terminating fatally a very marked phagocytosis is observed even in susceptible animals. The most typical example of this is furnished by swine erysipelas and mouse septicaemia. We know from the researches of Koch[438], followed by those of Loeffler[439], Schütz[440] and others, that in animals which have died from these two diseases the leucocytes are gorged with small specific bacilli. A method of vaccinating animals against the micro-organism of swine erysipelas was worked out by Pasteur and Thuillier[441] and was afterwards studied by many observers. Thanks to this method it has been possible to demonstrate the phenomena which may be observed in vaccinated animals (especially rabbits). Here also a phagocytosis takes place, even more rapid and more complete than in susceptible animals. What is more important, the intracellular digestion of the ingested bacilli is followed by the total destruction of the micro-organisms in the vaccinated animals, though in the normal animals this digestion is very imperfect.
The acquisition of immunity against micro-organisms is, therefore, due not only to the change from negative to positive chemiotaxis, but also to the perfecting of the phagocytic and digestive powers of the leucocytes—a general superactivity and adaptation of the phagocytic reaction of the immunised animal is produced. This conclusion, based upon a large number of well-established facts and in complete harmony with the whole of the data at our disposal concerning acquired immunity, has been attacked by Denys and Leclef[442] in their work on the streptococcus. They base their opposition upon experiments made in vitro on the action of serums and leucocytes on this micro-organism. They have compared the bactericidal power of mixtures of the serums of normal and of vaccinated rabbits with leucocytes isolated from exudations from these two groups of animals. The leucocytes, whether derived from normal or from vaccinated rabbits, when mixed with normal serum were equally incapable of ingesting and destroying the streptococci. When mixed with blood serum from vaccinated rabbits, however, the two kinds of leucocytes exhibited a very marked phagocytic reaction. Denys and Leclef conclude from this that phagocytosis, although an important factor in immunity, plays merely a secondary part and is dependent on the humoral properties. The experiments and views of these two observers have been generally received by the partisans of the bactericidal theory of the body fluids as an actual proof of this theory. We cannot agree. Researches extending over a long period have shown us that the study of phagocytosis in vitro can give only a very inexact and imperfect idea of the course of the phenomena in the living animal. Usually the leucocytes taken from the exudations, although amoeboid, no longer fulfil their phagocytic functions at a time when in the animal they would ingest micro-organisms with the greatest rapidity. As a general rule, existence outside the living body weakens them very considerably. But in some cases, rare it is true, the leucocytes although inactive in the animal exhibit intense phagocytosis when introduced into a hanging drop of fluid from an exudation or even of urine. In any case it is very hazardous to infer from phenomena which appear under these artificial conditions what takes place in the living animal. The value of the experiments of Denys and Leclef is still further marred by the fact that they mixed the leucocytes with blood serum. They appear to have lost sight of the fact that this fluid is far from corresponding to that which bathes the leucocytes in the living animal. The serums contain leucotoxin in greater or less quantity and it is not to be wondered at that the leucocytes when mixed with normal rabbit’s serum should perish very rapidly. Further, the serum of vaccinated rabbits is agglutinative (this fact, however, was not sufficiently elucidated in 1894 when the researches of Denys and Leclef were made) and the clumping of streptococci might simulate their destruction. In a word, the experiments of these observers have been carried out under such conditions that it is impossible to base upon them a refutation of data obtained in the living animal. Moreover, in the description of the phenomena which appear in the subcutaneous tissue of rabbits inoculated with the streptococcus, Denys and Leclef provide us with arguments against their own view.