Whilst, in natural immunity against micro-organisms, humoral phenomena play no prominent part, in acquired immunity these phenomena assume a much greater importance. The bactericidal power of the fluids of the body is, in natural immunity, reduced to a mere trace, for it has been demonstrated that the power of normal serums to destroy bacteria corresponds to no natural phenomenon of the living organism, but is dependent upon the presence of cytases which have escaped from the phagocytes at the time of the formation of the clot in vitro and separation of the serum. The presence of the fixative, that other important element in immunity, has been demonstrated in the normal fluids only in rare cases and in small quantity. The agglutinative property of these fluids has likewise shown itself to be little developed and without any importance in natural immunity.

In acquired immunity against micro-organisms, on the other hand, we find that the bactericidal and agglutinative powers of the fluids of the body are very greatly increased. With the discovery that the bactericidal property was so highly developed in the serums of animals that had been vaccinated against vibrios arose the belief in the acquisition of a new and purely humoral property. R. Pfeiffer, especially, insisted on the fundamental difference between the power of the serum of immunised animals to transform the cholera vibrios into granules and the corresponding property of normal serums. In the first case Pfeiffer’s phenomenon exhibited marked specificity; in the second, it was much more general. A normal serum transforms into granules, indifferently, vibrios that are very distinct from one another; whilst the serum of an animal vaccinated against a particular species or race of vibrios gives Pfeiffer’s phenomenon with this species or race only. Bordet’s^[364] researches have definitely settled this question. This investigator has shown that Pfeiffer’s phenomenon is produced, with all the usual serums, by means of the same substances, the cytases (alexine, or complement of Ehrlich). But in the serum of vaccinated animals there is added to these cytases the fixative (sensibilising substance of Bordet, immunising body or amboceptor of Ehrlich) which exhibits specific properties. Having thus carefully distinguished the two substances that set up the granular change in vibrios, Bordet shows that in vaccinated animals it is the fixative which increases in quantity, whilst the cytase remains pretty much in the same proportions as in the normal animal. He demonstrated, in fact, that when we take a very small dose of the serum of a vaccinated animal which by itself is incapable of transforming the vibrios into granules, about the same quantity of immunised serum or of normal serum must be added to it in order that Pfeiffer’s phenomenon may appear. The quantity of cytase, that soluble ferment which is necessary for the production of the phenomenon, is, therefore, about the same in the serum of a normal animal as in that of a well-vaccinated animal. Whilst the cytase does not increase as a result of vaccinal injections, the fixative, on the other hand, becomes more and more abundant. Consequently it is this second soluble ferment that impresses its characters on the blood serum and on some of the other fluids of the vaccinated animal. It has been pointed out in the preceding chapter that the fixative is found in the fluid of the oedema of vaccinated animals, although in less quantity than in their blood serum. It has also been mentioned that no fixative is found in the aqueous humour of well-vaccinated animals. It must be admitted that this ferment is not inseparably bound to the cells which produce it, as is the case with the cytases. I have already developed, at some length, the thesis that the cytases remain, in the normal animal, within the phagocytes, and only escape from them when these cells are destroyed, whether in the living animal (during phagolysis) or outside the animal (during the preparation of the serum). Gengou’s experiments with the plasma and the blood serum of normal animals have completely confirmed the fundamental observations that the cytases are not found free in the circulating blood. It is evident that the same law applies also to an animal that has acquired immunity. For this reason neither Pfeiffer’s phenomenon nor any analogous process that demands the action of cytases is ever produced in the anterior chamber of the eye, or in the subcutaneous tissue, or in oedema either active or passive. Further, it is in virtue of this same law that Pfeiffer’s phenomenon does not manifest itself even in the peritoneal cavity or in the blood vessels of vaccinated animals in which the phagocytes have been protected from phagolysis by previous injections of various fluids (physiological saline solution, broth, etc.). It would be very interesting to be able to demonstrate the absence of cytases in the fluids of immunised animals by experiments of the same order as those carried out by Gengou with the fluids of normal animals, but the obstacles to the realisation of this postulate are too great. We saw when discussing Gengou’s experiments that it is impossible to obtain in vitro a fluid identical with the plasma of living blood. The greatest precautions in collecting the blood and in its after treatment are insufficient to prevent coagulation taking place sooner or later. It follows that, as there is always a considerable quantity of free fixative in the plasma of immunised animals, an infinitesimal quantity of microcytase, set free from the leucocytes, is sufficient for the production of Pfeiffer’s or any other analogous phenomenon. There must be a great improvement in the methods of preparation of plasmas outside the body before it will be possible to undertake successful researches on the above problem. For the present we must rest satisfied with other proofs, already numerous and very demonstrative, of the absence of free cytases in the normal plasmas of vaccinated animals.

The cytases being found in about the same quantity and presenting the same properties in all animals that enjoy immunity whether natural or acquired, it must be the fixative which specially distinguishes these two categories of immunity. Now, the fixative is found in the serum of perhaps all cases of acquired immunity. Bordet and Gengou have studied it by the method already mentioned (Chap. VII.). A certain quantity of micro-organisms of various species is introduced into the serum. If the cytases, present in the serum when the experiment was commenced, ultimately disappear from it, it indicates that this ferment has been absorbed by the bacteria, thanks to the fixative, which consequently should be found in the serum under observation. The presence or absence of the cytases can be demonstrated by the production or absence of Pfeiffer’s phenomenon with vibrios.

The application of this method enabled Bordet and Gengou^[365] to satisfy themselves that the serum of animals immunised against several species of bacteria (plague bacillus, typhoid bacillus, bacillus of swine erysipelas, first anthrax vaccine, and Proteus vulgaris), really contains an appreciable quantity of fixative. It may, then, be accepted that the production of this substance is fairly constant in acquired immunity against bacteria, and that it constitutes one of the most important factors in such immunity.

The question has been raised: What is the nature of the substance to which the name of fixative is given? Pfeiffer and Proskauer^[366] have attempted to solve this question by making use of a serum which acts against the cholera vibrio and which they obtained by vaccinating animals with this vibrio. They carried out a long series of experiments which led them to the conclusion that this substance, which they term “cholera antibody,” cannot be identified with any of the albuminoid substances of the serum. Further, the fixative is not represented by any of the salts or extractive substances of the serum, because these substances dialyse easily, whereas the cholera antibody does not pass through the dialysing membrane. The fixative is wholly precipitated by alcohol, and is regarded by Pfeiffer and Proskauer as belonging to the category of soluble ferments, an opinion which is certainly shared by many other investigators.

What is it that communicates to this ferment its remarkably specific character? Without being able to give a precise answer to this question, the authors just cited point out the analogy that exists between the cholera antibody and the soluble ferments of yeasts which have been studied by Emil Fischer. Some of these act only upon certain special sugars in a manner equally specific. From a logical point of view it might be permissible to attribute the specificity of fixatives to something borrowed from the species of micro-organism that has played a part in their production. It has long been recognised that in old cultures of the cholera vibrio these micro-organisms are transformed into spherical granules, the arthrospores of Hueppe, which closely resemble the granules produced in Pfeiffer’s phenomenon. There are, then, undoubtedly, vibrionic products which act much as do the microcytases, and it would be very interesting if we could find them in the bactericidal ferments of the animal body. An attempt of this kind was undertaken by Emmerich and Löw^[367], who attribute the acquired immunity to a particular substance which they term “Nuclease-Immunproteïdin.” According to their hypothesis the microbial products which are produced in the animal during the period of vaccination—the nucleases—combine with proteid substances of the blood and organs to furnish the substance to which these authors have given such an elaborate name. In their most recent publication Emmerich and Löw even describe a method of producing this substance outside the animal body, by the action of ox blood, or better still pounded spleen, on the nuclease produced by the bacteria found in old cultures. To it they attribute the property of dissolving the various bacteria, of conferring immunity against and even of curing several infective diseases. But these authors do not say whether this remarkable substance is identical with, or analogous to, the antimicrobial ferments composed, as we have seen, of microcytase and fixative. It must be concluded that they look upon it as being similar to the alexine of Buchner, which is nothing more than a mixture of the two substances just named. Unfortunately the whole account given by Emmerich and Löw will do anything but gain over the reader, and in their publications no proof of their assertions can be found. Several of the facts advanced by them do not fall in with well-established data. Thus they speak of the complete lysis of the bacilli of swine erysipelas by their soluble “Erysipelase-Immunproteïdin” in vaccinated animals, a process that has never been demonstrated by them and which in no way accords with conscientious and carefully carried out observations. On the other hand, they cite facts which contradict one another. The “Pyocyanase-Immunproteïdin” is a substance which possesses an extraordinary bactericidal power, not only against the Bacillus pyocyaneus but also against several other bacteria, e.g. the bacilli of anthrax, diphtheria, typhoid, and plague. This substance rapidly breaks up these bacteria, and cures diphtheria and experimental anthrax. But it is, at the same time, so affected by the invasion of the most common bacteria, such as Bacillus subtilis, that it is necessary to add antiseptics in order to preserve it. To these contradictions, inaccuracies, and uncertainties must be added further the advice, given by Emmerich and Löw to bacteriologists, not to attempt to reproduce their experiments, because they may easily fail, and I think that, in spite of the seductiveness of the attempt to attribute to bacterial products a share in the elaboration of antimicrobial substances, we must conclude not to follow these authors further. It is better to confess our ignorance of the chemical composition of these substances in general and of the fixatives in particular.

As the fixatives resist temperatures much higher than those which destroy the cytases, in this respect resembling the agglutinative substances so frequently found in the fluids of vaccinated animals, there has long been a tendency to identify them with these latter. It is indisputable that between the fixatives and the agglutinative substances the analogies are fairly numerous. Both are produced in quantity during the process of immunisation, and are found not only in the blood serum but also in the fluids of the living animal, especially in the fluids of exudations and transudations. Both dialyse through parchment more readily than do the cytases. Buchner^[368] has demonstrated that his alexines (bactericidal substances of normal serum) will dialyse only where the lower fluid is pure water; dialysis is nil when the distilled water is replaced by physiological saline solution. The fixatives and agglutinins, as demonstrated by Gengou^[369] for the latter, pass almost completely through the dialyser in the case of pure water, and one-half still passes when the lower fluid approaches as nearly as possible to normal serum.

In spite of these analogies, however, the agglutinative property must be sharply distinguished from the fixative power of serums. In this fluid, derived from normal animals, the agglutinative property is often very marked when the power of fixing the cytases is totally, or in great part absent. Bordet and Gengou^[370] have demonstrated also that feebly agglutinative serums of persons convalescent from typhoid fever may exhibit a great capacity for fixing the cytases. Other facts, to be mentioned later, confirm the real difference between the fixative and the agglutinative properties.

The agglutination of bacteria was noted during the course of a series of researches on the acquired properties of the blood serum of vaccinated animals. Charrin and Roger^[371], seeking to obtain a clear idea of the difference between the serum of normal animals and that of animals vaccinated against the Bacillus pyocyaneus, observed that this bacillus developed in the normal fashion in the former, but in the latter gave rise to special forms of growth. Instead of growing in the form of rods, it elongates into segmented filaments which become entangled and fall to the bottom of the tubes, leaving a supernatant limpid serum. I was able not only to confirm the accuracy of this observation for the Bacillus pyocyaneus, but to extend it to Gamaleia’s vibrio and to the pneumococcus^[372]. In all these instances we have a modification of the bacteria developed in specific serums coming from vaccinated animals. Later, Bordet^[373], during his researches on the bacteriolysis of vibrios in vitro, observed that these vibrios, when introduced into the blood serum of vaccinated animals, lose their movements and soon unite into more or less voluminous masses. This observation was confirmed by Gruber and Durham^[374], who were the first to apply it in the specific diagnosis of bacteria. They showed that the agglutinating power of vaccinated animals, although not rigorously specific, might, nevertheless, be utilised for the differentiation of certain bacteria, especially the cholera vibrio and the typhoid bacillus. But, independently of this result, Gruber^[375] essayed to formulate a theory of acquired immunity based on the agglutinative property of the serum. He accepted, in connection with the phenomenon of the destruction of the bacteria, Bordet’s hypothesis of the concurrent action of two substances, of which one, the bactericidal substance proper, is nothing but the alexine of Buchner, the second being that which agglutinates the bacteria. This agglutination, according to Gruber, results from the swelling of the bacterial membrane which becomes viscous and so leads to the cohesion of the bacteria and the formation of clumps. Thus transformed and rendered motionless, the bacteria succumb more readily to the destructive action of the alexine. It is supposed that the phagocytes do not intervene at all in these cases of acquired immunity, except in a purely secondary fashion when they ingest the bacteria already greatly weakened by the united action of the agglutinin and the alexine. The principal rôle in this theory of immunity is thus given to the agglutinative substance, which is regarded as being a microbial product, modified by the macrophages and thrown into the blood.

The discovery of this agglutination of bacteria has acquired great importance, especially in connection with its application to the diagnosis of typhoid fever. Widal^[376] succeeded in showing that typhoid bacilli agglutinate readily under the influence of blood serum and other fluids (milk, transudations, tears, etc.) derived from patients suffering from typhoid fever. As this phenomenon could be utilised for the early recognition of the disease, it began to be studied with great care and many interesting data concerning it have been collected. The general outcome of these researches accords with the conclusions drawn by Widal, and the serum-diagnosis of typhoid fever has taken an important place among the methods used for the recognition of this disease. This aspect of the question, however, does not interest us from the point of view of the problem of immunity which we now have under consideration, and we cannot here enter upon the study of the serum-diagnosis of typhoid fever and certain other diseases (cholera, tuberculosis, pneumonia). Moreover, we must refrain from any analysis of the hypotheses advanced to explain the mechanism of agglutination. A lively discussion has been carried on between the partisans of the chemical theory—according to whom the agglutinin acts directly on the agglutinable substance of the bacteria—and the advocates of the physical theory, led by Bordet^[377], who attribute the agglutination to modifications in the molecular attractions which unite the agglutinable elements, be it between each other or with the surrounding fluid. At one time it was thought that Roger’s^[378] observation that the cell membranes of Oïdium albicans, when cultivated in the specific serum of immunised animals, increased in volume and became greatly swollen, settled the question in favour of Gruber’s theory. But the objection formulated by Kraus and Seng^[379], on the one hand, and by Bordet, on the other, dealt a severe blow to this view. As the serum employed by Roger was not deprived of its cytases (alexine), the viscosity of the membrane of the fungus could not be attributed to the agglutinin. When Bordet^[380] demonstrated that the red blood corpuscles, under the influence of the serums, undergo an agglutination as marked as that seen in bacteria, it enabled us to study this phenomenon in the large red corpuscles of birds, in which no one has ever been able to demonstrate any viscosity of the corpuscular stroma. In a mixture of red corpuscles of bird and mammal, submitted to the action of a serum which agglutinates the former only, the red corpuscles of the mammal never unite with those of the bird, although this should undoubtedly take place if the membrane of the agglutinated corpuscles had really become viscous. All the facts collected up to the present are, therefore, in favour of Bordet’s physical theory in which an analogy between the phenomena of agglutination and of coagulation is traced.

The point that interests us more particularly in regard to agglutination is the relation of this phenomenon to immunity. We have already given (Chapter VII) the arguments which render it impossible for us to attribute to the agglutinative property of the fluids of the body any rôle, however unimportant, in natural immunity against micro-organisms. We must now study the importance of this property in the condition of acquired immunity, in which the agglutination of micro-organisms by the fluids of the body is much more frequent and active than in natural immunity.

The first question to be settled is the following: Is the agglutinative property really constantly present in the fluids of vaccinated animals? The blood serum of animals that have acquired immunity is unquestionably usually agglutinative as regards the corresponding micro-organism. This agglutination may be more or less pronounced, but it certainly exists in the great majority of cases. Nevertheless, examples can be cited in which, in spite of the refractory condition acquired as the result of immunisation, the serum exhibits not a trace of agglutinative power. Having demonstrated that several bacteria (Bacillus pyocyaneus, Diplococcus pneumoniae, Vibrio metchnikovi) develop in the serum of vaccinated animals in the form of elongated filaments more or less interlaced, I was quite prepared to allow that this fact might be of general import. But the study of a cocco-bacillus which produces the pneumo-enteritis of swine and which was isolated by Chantemesse during an epizootic at Gentilly, led me to believe that this was not the case. As this bacillus is characterised by great motility, I concluded^[381] that it was identical with that of the hog cholera of American writers. Theobald Smith^[382], to whom I sent a specimen and who is a competent authority on this question, refers it, however, to the species which produces swine plague. Knowing that the question of these two bacteria is not finally settled, it is impossible to come to an absolute decision in the matter. Fortunately, from the point of view of immunity, this is of no great importance. The point upon which I must lay stress is that the serum of rabbits vaccinated against the Gentilly bacillus, when sown with this cocco-bacillus, gave very abundant and uniformly turbid growths. In my researches, undertaken at a period when the rapid agglutination of micro-organisms added directly to the specific serum had not yet been recognised, I noted merely that the cocco-bacilli which grew in the blood serum of vaccinated rabbits presented their normal form and gave rise to a general turbidity of the fluid. Since then, however, it has often been observed that the mode of development of a micro-organism in a serum gives an even more delicate indication than does the agglutination properly so called, produced by the serum to which has been added an organism cultivated on its usual medium. Thus Pfaundler^[383] saw that Bacillus coli and Proteus vulgaris, which were not agglutinated by certain serums, developed in them in an unusual fashion and produced very long and interlacing filaments. When a serum is incapable of revealing its properties by agglutinative reaction properly so called, it is sown with the corresponding micro-organism and the development is then compared with that observed in a normal serum. Frequently a very marked difference is noted, the same organism growing into filaments in the specific serum and forming rods only in the normal serum. The first mode of development is sometimes designated “Pfaundler’s reaction.”

In the serum of rabbits vaccinated against the Gentilly cocco-bacillus, no filaments corresponding to those met with in the agglutinative reaction are formed, but bacilli are produced. In spite of this the animals that furnish the serum show a distinct resistance to infection. More recently, Karlinski^[384] has studied the properties of the serums of animals treated with the cocco-bacilli of hog cholera and swine plague. He was able to demonstrate that blood serum from oxen that had received repeated injections of cultures or toxin of hog cholera, was not only incapable of killing the cocco-bacilli of the two swine diseases but it even “gave rise to no agglutination” of the two bacilli and did not arrest the motions of those of hog cholera. On the other hand, serums have been obtained from other species of animals (dog, pig) which brought about the typical agglutination of the cocco-bacillus of hog cholera^[385].

In the preceding chapter, Gengou’s experiment on the serum of a dog that had been treated with a virulent culture of anthrax has already been cited. This serum did not agglutinate the bacillus, even of the first vaccine of Pasteur. Nevertheless, a second dog treated with an attenuated culture of this bacillus furnished an agglutinative serum. The immunisation of the first dog was carried very much further than that of the second, but the agglutinative properties were in inverse order. Sawtchenko, in his study of immunity against anthrax, demonstrated that the subcutaneous exudation from vaccinated rats does not agglutinate the bacillus which usually exhibits such a great tendency to collect into clumps.

Agglutination has been studied particularly carefully in typhoid fever. We know that after an attack of this disease, an acquired refractory condition is produced which lasts for a considerable period. In most cases the agglutinative power of the blood diminishes very rapidly, and disappears a few weeks after the commencement of convalescence. It is only in rare cases that it persists for years^[386]. On the other hand, during the period of apyrexia which precedes the relapse in typhoid fever and during the period of relapse, the agglutinative power may manifest itself in a very marked degree. In an observation made on a case reported by Widal and Sicard^[387], the agglutinative power was raised, two days before the relapse, to a ratio (1 : 150) it had never attained during the first attack. “The appearance of the relapse, two days after this observation”—these authors add—“renders it evident that the agglutinating reaction is independent of the state of immunisation.” Analogous cases have been pointed out repeatedly by several observers.

The examples cited show, on the one hand, that the serum of individuals endowed with acquired immunity may be without any agglutinative property, but, on the other, that this power may be highly developed in the serum of susceptible individuals. The argument based on these data may be corroborated by several other series of facts. Thus, Salimbeni^[388] has pointed out that the cholera vibrio is not agglutinated in the fluids of immunised animals. The subcutaneous exudation of a horse treated with a large quantity of these vibrios does not agglutinate Koch’s vibrio except outside the body. When this exudation is drawn off shortly after the injection of the vibrios, the organisms render the fluid uniformly turbid. But a short exposure to the air is sufficient to bring about the agglutination of the vibrios in the same exudation. Guided by this observation, Salimbeni carried out comparative experiments on the action of the serum of vaccinated animals outside the body, in tubes deprived of oxygen and in others exposed to the air. In the former agglutination did not take place or was very incomplete, in the latter it soon came on. This fact accords perfectly with the observation of Pfeiffer’s phenomenon in the peritoneal cavity of guinea-pigs from which we withdraw a fluid containing granules that have resulted from perfectly isolated vibrios. In other micro-organisms a difference has been noted in this respect. Thus Gheorghiewsky has seen the agglutination of the Bacillus pyocyaneus produced under the influence of the serum of vaccinated animals, even in tubes deprived of oxygen. Durham has made a similar observation in the case of the typhoid bacillus. When, however, Trumpp^[389] wished to satisfy himself as to the agglutination of the same organism in the body of well-vaccinated guinea-pigs, he obtained only imperfect results. He concluded from his experiments “that the formation of typhoid clumps may precede the breaking down of the bacteria in the animal body itself, but only under certain conditions—when the degree of immunity of the animal is sufficiently high and when the bacilli introduced are not too numerous” (p. 130). In the case of the typhoid bacillus, a certain degree of agglutination is produced inside the animal body, but it is markedly increased in the fluids that have been withdrawn and exposed to the action of the air.

It has been demonstrated, repeatedly, that the agglutination of micro-organisms by their specific serums does not prevent their growth and multiplication. These agglutinated organisms lose none of their virulence. Issaeff^[390], working in my laboratory, carried out an investigation on this point in the case of the pneumococcus. He vaccinated rabbits against this organism and satisfied himself that the organism still grows well in the blood serum of such rabbits; but, instead of presenting the typical form of lanceolate diplococci, the pneumococcus, under these conditions, forms very long chains of true streptococci. Having filtered the cultures in order to get rid of the serum, he injected them into rabbits and mice and demonstrated that the pneumococci had retained to the full their initial virulence. Sanarelli^[391] carried out corresponding experiments with Gamaleia’s vibrio, which, as we know, also forms chains in the serum of vaccinated animals. When filtered on a paper filter and washed with physiological saline solution, the vibrios were found to be just as virulent as were the control vibrios grown in the serum of susceptible animals. More recently, Mesnil^[392] demonstrated the same point in connection with the bacillus of swine erysipelas. He experimented on cultures that were agglutinated after their formation and also on others agglutinated as they were growing. The fluid of the culture was decanted and replaced by fresh broth until the elimination of the serum was complete. Mice, inoculated with the washed clumps, died in the normal period, thus affording proof that “agglutination in no way alters the vitality and virulence of the bacillus of swine erysipelas” (p. 492).

We can readily understand, after the demonstration of these various facts, that it is impossible to maintain Max Gruber’s theory that the agglutinative power constitutes the fundamental basis of acquired immunity. Hence this writer, after publishing several preliminary notes in 1896, has not yet decided to give to his hypothesis a more extended development. Nor has any one else attempted to defend it.

It is probable that in certain special cases the immobilisation of very motile bacteria and their agglutination into clumps may facilitate the reaction of the animal organism, especially the rapidity of phagocytosis. Thus, Besredka^[393] observed that guinea-pigs when inoculated with typhoid bacilli that had previously been mixed with the blood serum of normal animals survived. The most active amongst these serums was ox serum heated to 60° C. Guinea-pigs furnished a serum which was much less active. The resistance of guinea-pigs, inoculated into the peritoneal cavity, was in direct ratio to the agglutinated condition of the bacilli. Besredka lays stress on the facility with which the bacilli, when agglomerated into large clumps, were ingested by the phagocytes, and suggests that there is a certain stimulating action of the serums on the leucocytes. When he injected into guinea-pigs a mixture of typhoid bacilli and guinea-pig’s serum, made immediately before injection, his animals died from infection. But when he left the bacilli for some time in contact with the guinea-pig’s serum outside the body, and did not inject the mixture until after agglutination was complete, the inoculated animals usually survived. This experiment indicates the part played by agglutination in the resistance offered by the animal, and at the same time proves that in the body of the guinea-pig the agglomeration of the micro-organisms into clumps does not take place to the same degree as in the serum prepared in, and left in contact with, the air.

In any case, the data collected by Besredka cannot be put forward as an argument in favour of the essential part played by agglutination in acquired immunity, nor can they weaken the facts indicated as to the absence of agglutinative power in examples of acquired immunity and as to the virulence of the agglutinated micro-organisms. The part played by agglutination in this immunity is merely accidental and subordinate.

Special researches have been carried out with the object of defining, exactly, the origin of agglutinins in the body of an animal that has acquired immunity. Observers are unanimous in recognising that, of all parts of the organism, the blood is richest in agglutinin. This substance is found in the blood serum as well as in the plasma. From this (corroborated by the agglutinative property of other fluids, such as the pericardial fluid, oedemas very poor in formed elements, etc.) it follows that the agglutinin circulates in the blood and lymph of the living animal. Several observers, amongst whom I may cite Achard and Bensaude^[394], Arloing^[395], and Widal and Sicard^[396], put to themselves the question whether, before passing into the blood, the agglutinin is not formed in the exudation developed at the seat of inoculation of the micro-organisms. Their conclusions were invariably negative; they were never able to find more agglutinins in these exudations than in the blood. Pfeiffer and Marx^[397] had occasionally observed that their animals, inoculated with the cholera vibrio, early exhibited an agglutinative power in the spleen; but this result was not met with sufficiently constantly to enable them to draw a positive conclusion. A little later, van Emden^[398] studied in detail the distribution of the agglutinative property in the body of an animal inoculated with Bacillus aërogenes. His researches led him to the conclusion that the spleen and the lymphoid organs must be regarded as the source of the agglutinins. Shortly after the inoculation of the bacilli, an extract of the spleen was more agglutinative than the blood or any of the other organs. In rabbits from which the spleen had been removed, the same rôle was filled by the bone marrow and probably also by the lymphatic nodules. But this preponderance of the haematopoietic organs did not continue long, the blood soon becoming the most important seat of the agglutinative power.

The proof that this question of the origin of the agglutinins is a very delicate and difficult one is afforded by an investigation very carefully carried out by Gengou^[399] on the agglutination of the attenuated anthrax bacillus (Pasteur’s first vaccine) by the fluids and organs of normal and prepared guinea-pigs. This observer was never able to obtain any confirmation of the results obtained by van Emden with another micro-organism. In Gengou’s guinea-pigs it was always the blood fluid which showed itself most agglutinative, the organs exhibiting merely a feeble and inconstant agglutinative power. As the extracts of leucocytes were always found to be markedly less active than the blood and the fluids of the exudations, Gengou was obliged to come to the conclusion that the agglutinins cannot be regarded as products of the cells of the animal body; this he sums up by saying that “in the increase of the agglutinative power of its blood the organism of the animal plays only a relatively passive part” (p. 337).

I think that, in spite of the facts established by Gengou, his conclusion can scarcely be regarded as final. The agglutinative property, developing in the animal body, must be attributed to some cellular influence, because we know that the prolonged sojourn of micro-organisms in the animal fluids is incapable of conferring on them this power. As Gengou’s experiments did not permit him to attribute the formation of agglutinin to any formed element, it must be concluded that, although perfectly exact, they were insufficient to solve the problem. Gengou killed his animals at a stage when their blood was already pretty strongly agglutinative. At this stage the organs only possessed it to a much more feeble degree. Perhaps, if he had examined his animals at an earlier stage, when the blood possessed a much less marked agglutinative power, he might have obtained a more powerful agglutination with an extract of the organs. In my researches on the resorption of cells, I observed, on several occasions, that the abdominal fluid of guinea-pigs which had received an injection of goose’s blood became agglutinative before the blood serum. Later, however, the blood exhibited a greater agglutinative power than did the peritoneal fluid. If to this fact we add the results of van Emden’s experiments, we shall be tempted to assign to the cells found in the peritoneal exudation and in the lymphoid organs a share in the production of the agglutinin. This question of the origin of the agglutinative power is, however, a very difficult one, and it is impossible, in the imperfect state of our knowledge, to express oneself in a more positive fashion. Fortunately, according to the whole of our data on this phenomenon, the part played by agglutination in immunity can only be very inconsiderable, and we may be allowed to consider our general problem without concerning ourselves over much about the origin of the agglutinative property.

Among the definite results obtained from the study of the agglutinins, it may be specially pointed out that these substances can in no way be identified with the fixatives. These latter were, for long, spoken of as preventive substances. They are so termed in the early papers of Jules Bordet treating upon this question. The explanation of this designation is that, for a series of years, the presence of the fixatives was revealed chiefly by the preventive or protective property of the media which contained them.

To gain a clear conception of this protective property, which occupies so important a place in the study of acquired immunity, we must go back to an epoch in our science when it was sought to prove that the fluids of the body played a part in the production of immunity. Shortly after the earliest researches on the bactericidal power of the blood had been made, the idea of applying the results obtained in this direction to the production of immunity in animals by means of injections of blood occurred. The first step in this direction was taken by Richet and Héricourt^[400], who succeeded in vaccinating rabbits against a variety of staphylococcus by means of defibrinated dog’s blood. The dog is naturally refractory against this organism, and the blood of a normal dog exercised a certain vaccinal or protective influence on rabbits inoculated with the staphylococcus. But this action was much more marked when Richet and Héricourt employed the defibrinated blood of dogs which had previously received inoculations of the staphylococcus. Shortly after this observation, von Behring^[401] made his discovery of antitoxins in the blood serum of animals immunised against tetanus and diphtheria toxins. In collaboration with Kitasato he demonstrated that the serum of these animals, when injected into normal animals, protected them against intoxication by the poisons of diphtheria and tetanus. This great discovery, which has been confirmed on all sides and extended to other poisons, gave rise to the view that a serum exerting any protective power depends solely on its property of impairing the action of the toxins. A more careful study of the phenomena which appear under the influence of the serums has, however, demonstrated the inaccuracy of this view. I was able to furnish the proof^[402] that the blood serum of rabbits vaccinated against the micro-organism of the Gentilly pneumo-enteritis prevented normal rabbits from contracting a fatal infection. Nevertheless, the serum exerted no influence on the toxin of this micro-organism; the rabbits that received the minimal lethal dose of this toxin, mixed with serum from vaccinated rabbits, died, as did the control animals, from rapid poisoning. It was evident then that this serum, which prevented infection without in any way hindering intoxication, could not be classed in the category of antitoxic serums. We find ourselves, therefore, in the presence of a new property of the fluids of the body to which we have given the name of protective or anti-infective power. We are driven to this conclusion the more as the serum in question was neither bactericidal nor agglutinative.