CHAPTER VIII

SURVEY OF THE FACTS BEARING ON ACQUIRED IMMUNITY AGAINST MICRO-ORGANISMS

The discovery of attenuated viruses and its application to vaccination against infective diseases.—Vaccination by microbial products.—Vaccination with serums.—The acquired immunity of the frog against pyocyanic disease.—The acquired immunity against vibrios.—Extracellular destruction of the cholera vibrio.—Part played by two substances in Pfeiffer’s phenomenon.—Specificity of fixatives.—Phagolysis and its relation to the extracellular destruction of vibrios.—Part played by phagocytosis in the acquired immunity against vibrios.—Fate of the spirilla of recurrent fever in the organism of immunised guinea-pigs.—Acquired immunity against the bacteria of typhoid fever and pyocyanic disease.—Acquired immunity against swine erysipelas and anthrax.—Acquired immunity against the streptococcus.—The acquired immunity of rats against _Trypanosoma_.

[Sidenote: [218]]

Certain of the hypotheses on acquired immunity are of as ancient origin as are those on natural immunity. For example, it has for long been known that man is constitutionally refractory to certain diseases which are very fatal to cattle. It has also been recognised that after a first attack of a contagious disease, such as small-pox, measles, scarlatina, typhoid fever, etc., man acquires a lasting immunity; and that the same rule applies to domestic animals, for example, cattle that have recovered from cattle plague or sheep that have got better from sheep-pox, become refractory against these diseases.

[Sidenote: [219]]

The discoveries of variolisation and vaccination, as methods of conferring on man a resistance to small-pox, have notably advanced our knowledge upon acquired immunity. The researches on the properties of vaccine had already led to some important results. But it is only since the publication of Pasteur’s investigation, carried out with his collaborators Chamberland and Roux, in the first place, and with Thuillier later, that we have been able to take up the study of acquired immunity in a really scientific manner. The first in this series of discoveries, which have opened up a path so fruitful to science and medical art, was the discovery of the attenuation of micro-organisms. The small cocco-bacillus of fowl cholera after several weeks’ culture in broth was found to have become markedly attenuated in virulence. To Pasteur the idea occurred of testing whether fowls that had resisted the inoculation of these attenuated organisms had acquired any real immunity against virulent fowl cholera. Experiment confirmed his expectation and led to the discovery of the vaccine against this disease. The method was at once applied to other infective epizootic diseases and shortly afterwards Pasteur, Chamberland and Roux found a method of preserving sheep and cattle from anthrax. To attain this end it was found necessary to prevent the bacillus from producing spores (in this they succeeded by cultivating it in broth at a temperature of 42°·5 C.), because these spores fix the virulence and prevent attenuation. Having overcome this main obstacle, Pasteur and his collaborators found that their cultures, thus deprived of spores, become attenuated on exposure to the air and so become transformed into vaccines. They were thus able to prepare their two anthrax vaccines which soon found such wide application in practice. A few years later, Pasteur and Thuillier discovered the vaccines against swine erysipelas and, in collaboration with Roux and Grancher, Pasteur made the first application of his discoveries to the vaccination of man against rabies.

[Sidenote: [220]]

The path thus opened up was traversed by many other investigators and led to many very remarkable discoveries. Vaccination with micro-organisms became a recognised method and in the hands of Arloing, Cornevin and Thomas, soon found its application to symptomatic anthrax. The next step in this onward progress of science was taken when Salmon and Smith, working at hog-cholera, demonstrated the possibility of vaccinating not only with hog-cholera bacilli, but also with culture fluids in which these organisms had developed. These fluids, when completely deprived of micro-organisms by filtration, protected the experimental animals from virulent hog-cholera. This discovery, at first sceptically received, was soon confirmed and extended by the work of other investigators. Beumer and Peiper extended it to the experimental disease set up by the typhoid bacillus in small laboratory animals; Charrin applied it to the disease that he produced by means of the bacillus of blue pus; and Chamberland and Roux prepared vaccines from the soluble products of the septic vibrio and of the bacillus of symptomatic anthrax. And now, as the result of these investigations, vaccinations by microbial products are in everyday use in all research laboratories. The vaccinations now used (anthrax, symptomatic anthrax, swine erysipelas and rabies) are still being carried out by means of the inoculation of living viruses.

The comparative history of acquired immunity is still very incomplete. The facts known concerning the adaptation of unicellular organisms to all kinds of injurious influences of a physical or chemical nature enable us to perceive that acquired immunity is just as general in living beings as is natural immunity; but it is impossible, in the present state of our knowledge, to confirm this hypothesis by exact and experimental data. The reason for this lies in the great difficulty we have in carrying out experiments on the lower animals. The majority of the Invertebrata in captivity do not remain alive long enough and can not be sufficiently often inoculated for us to obtain in them a well marked acquired immunity against micro-organisms. Kowalevsky[306], the celebrated Russian zoologist, has tried to overcome these various difficulties by making use of Myriapods. He found first that _Scolopendrae_, when inoculated with anthrax bacilli, die therefrom during the heats of summer, the blood containing a number of anthrax bacilli. But when the temperature does not exceed 17°–18° C., a fairly large number of these myriapods survive. The same survival was observed when Pasteur’s first vaccine was injected. Kowalevsky utilised the _Scolopendrae_ that had resisted the first injection of anthrax bacilli to ascertain whether they had contracted an acquired immunity. The results were not absolutely demonstrative and Kowalevsky sums up his results in the following words: “I cannot say, therefore, that I have succeeded in solving this question of vaccination, but it appears to me very probable” (p. 607).

[Sidenote: [221]]

In view of this doubt, I asked Mesnil to make a fresh attempt, making use of _Scolopendrae_ and inoculating them with anthrax bacilli. These creatures, however, were so delicate and so little capable of remaining alive under the artificial conditions of their captivity, that the attempt soon had to be abandoned. I tried to obtain better results with the larvae of _Oryctes nasicornis_; here again the difficulties were too great. These insects exhibit a perfect natural immunity against certain micro-organisms, but for others they showed an insurmountable susceptibility. It is very evident, then, that it is not an easy matter to set up an acquired immunity in the Invertebrata.

It was necessary, therefore, to go higher up the animal scale and have recourse to cold-blooded vertebrates. The choice naturally fell on the frog. I asked Dr Gheorghiewski[307], who was working in my laboratory, to try to vaccinate the Batrachians against pyocyanic disease. I ought first to state that the bacillus of blue pus is pathogenic for the frog, which it kills both at the ordinary laboratory temperature, and at that of the incubator, 30°–37° C. In the first case the fatal dose is much greater than in the second, but it is always easy to induce a fatal infection. In this respect, therefore, the _Bacillus pyocyaneus_ is much better adapted for study than the anthrax bacillus or many other micro-organisms. Gheorghiewski vaccinated green frogs (_Rana esculenta_), which had been accustomed to the incubator temperature, 30° C., by injecting every 4 to 7 days considerable doses of cultures of _Bacillus pyocyaneus_ heated to 80° C. in order to kill all the micro-organisms. Some (3–4) weeks afterwards the prepared frogs became more resistant to the _Bacillus pyocyaneus_ than were the controls placed under the same conditions. The frogs, inoculated with a fatal dose of the bacilli, clearly exhibited a certain, though slight, degree of acquired immunity. They withstood a dose that was always fatal to the controls or even a dose and a half, but died when injected with double the fatal dose. The lymphatic fluid of the vaccinated frogs feebly agglutinated (1 : 20–1 : 30) the _Bacillus pyocyaneus_ although it still formed an excellent culture medium for this organism. Gheorghiewski satisfied himself that the agglutination was insufficient to assure immunity to the frog. The bacilli agglutinated into clumps were very virulent.

[Sidenote: [222]]

A detailed examination of the phenomena observed in the immunised frogs revealed the following facts. During the earliest stage the bacilli, injected into the dorsal lymphatic sac, were found free in the fluid, retained their form and were not transformed into granules. The bacilli carried by the lymphatic current spread rapidly throughout the body. Very shortly after inoculation, however, some of the leucocytes began to ingest the bacilli which became transformed into spherules within these cells. Later, the phagocytic reaction increased and at the end of 15 to 20 hours all the bacilli were found inside leucocytes. It was easy to demonstrate that these bacilli had been ingested in a living condition. Forty-eight hours after inoculation, no bacilli were to be found in the lymph of the dorsal sac, either inside or outside the cells. But this fluid when sown on nutrient media gave colonies of the _Bacillus pyocyaneus_ up to 15 and even 18 days after inoculation.

We may conclude from these facts that the cold-blooded vertebrata are capable of acquiring immunity to a slight degree and that, in this acquired immunity, a marked phagocytosis may be observed, but no bactericidal action of the fluids.

In order to gain a more complete idea of the mechanism of acquired immunity, it is necessary to observe it in higher vertebrates in which a well developed immunity of this type is readily obtained. Here we must have recourse to mammals and pass in review an ample number of examples, before we attempt to give to our readers a general summary of the question.

For long, researches on acquired immunity were confined almost exclusively to the analysis of the facts observed in animals submitted to anti-anthrax vaccinations by means of the two vaccines of Pasteur. A large number of important facts were thus collected, the more weighty of which must be presented to the reader. But, before entering on the subject, a general orientation on acquired immunity in laboratory animals against vibrios is indispensable as this example dominates, so to speak, the whole of the chapter on acquired immunity against micro-organisms.

[Sidenote: [223]]

Von Behring and Nissen[308], in their researches on the bactericidal power of serums, examined, amongst others, several specimens of serums coming from animals that had been vaccinated against various micro-organisms. In the majority of the examples given by them the acquired immunity produced no increase in this power, but the blood serum of guinea-pigs that had been immunised against Gamaleia’s vibrio (_Vibrio metchnikovi_) was found to be much more bactericidal as regards this micro-organism than the serum of normal susceptible guinea-pigs. These authors came to the conclusion that in acquired immunity, at least as regards the vibrio mentioned, the chief part is played by a bactericidal substance which is developed in the fluids of the vaccinated animals. They were content with the mere demonstration of this fact without making any attempt to follow the course of events in the destruction of the vibrios as it occurs in the organism of the vaccinated guinea-pig. R. Pfeiffer[309] in collaboration with Issaeff sought to fill this gap. But, instead of taking Gamaleia’s vibrio, these observers concentrated their attention on the study of the acquired immunity of guinea-pigs against the cholera vibrio. As this vibrio is as a rule less virulent than Gamaleia’s vibrio, it was necessary, in order to obtain a fatal infection, to inject it, not into the subcutaneous tissue but into the peritoneal cavity. We have already seen (Chapter VI) that the cholera vibrio when inoculated into the peritoneal cavity of the guinea-pig, there meets with a vigorous resistance on the part of the leucocytes which seize the living and virulent vibrios and digesting them rid the animal of their presence. But when the dose of the vibrios is increased, they multiply in spite of the phagocytic reaction; they are found swarming in the peritoneal cavity, whence they invade the lymphatic and blood vessels and cause the death of the animal. It is easy, then, to induce a fatal infection of the guinea-pig with the cholera vibrio. But it is also easy to vaccinate these animals against this experimental disease. We have only to inoculate them with a non-fatal quantity of living cholera vibrios, or to inject into them a culture in which the vibrios have been killed by heat, or some of the culture fluid from which the vibrios have been removed by filtration. All these methods soon produce an acquired immunity in guinea-pigs. If, when this has been brought about, a little blood is withdrawn and to the serum a small quantity of cholera vibrios is added, _in vitro_, we can readily demonstrate their disappearance, under the influence of the bactericidal substance dissolved in the fluid. In this respect there is, then, a marked analogy with the fact established by v. Behring and Nissen as regards Gamaleia’s vibrio.

[Sidenote: [224]]

When into the peritoneal cavity of vaccinated guinea-pigs a certain quantity of cholera culture containing virulent and very motile vibrios is injected, we find that in the peritoneal fluid drawn off by means of a fine pipette, the vibrios have undergone profound changes in the refractory organism. Even a few minutes after the injection of the vibrios, the leucocytes disappear almost completely from the peritoneal fluid; and only a few small lymphocytes and a large number of vibrios, the majority of which are already transformed into granules, are found (fig. 39); and there is presented a most typical case of Pfeiffer’s phenomenon. Alongside the round granules may be seen swollen vibrios, and others which have kept their normal form, but all are absolutely motionless. Some of these granules are gathered into small clumps, others remain isolated in the fluid. When to the hanging drop containing these transformed vibrios a small quantity of a dilute aqueous solution of methylene blue is added, we observe that certain granules stain very deeply, whilst others take on merely a very pale tint, scarcely visible. Many of these granules are still alive, because it is easy to watch them develop outside the animal and elongate into new vibrios. A large number of the granules, however, no longer exhibit any sign of life and are evidently dead. R. Pfeiffer and certain other observers affirm that the granules may be completely dissolved in the peritoneal fluid just as a piece of sugar dissolves in water. We have repeatedly sought for this disappearance of the granules in hanging drops of the peritoneal fluid, without being able to find any diminution in the number of these transformed vibrios, even after several days; nor have we been able to observe the phenomenon of the solution of the granules. It is at any rate indisputable that this granular transformation is a manifestation of very profound lesions undergone by the cholera vibrios under the influence of the peritoneal fluid of the immunised animal.

[Illustration:

FIG. 39. Cholera vibrios in the peritoneal cavity showing Pfeiffer’s phenomenon. ]

[Sidenote: [225]]

An attempt has been made to define the mechanism of Pfeiffer’s phenomenon more exactly, and Fischer[310] has sought to refer it to osmotic action, exercised by the salts of the fluids in which the vibrios are suspended. These vibrios, under the action of media richer or poorer in salts than is the fluid in which they had developed, are said to present an increase of their internal pressure, in consequence of which the vibrios swell up or allow a spherical droplet of protoplasm to escape at one of their poles. This explanation was inadequately supported by its author and cannot be regarded as proved. On the other hand, one is compelled to the conclusion that the granular transformation is due, as we shall see later, to a fermentative action of the peritoneal exudation.

Whilst the vibrios are undergoing this transformation in the peritoneal cavity of an immunised guinea-pig, the animal recovers from a _malaise_ that is quite transitory and continues to live, whilst normal unvaccinated guinea-pigs die, an enormous quantity of vibrios swarming in the peritoneal exudation. The difference between the two animals is most striking, and we can readily understand that Pfeiffer was so impressed by it that he was led to attribute the acquired immunity of his guinea-pigs solely to the granular transformation set up by a bactericidal substance contained in the fluids of the immunised animals.

[Sidenote: [226]]

The ease with which we can gain an idea of the change of form in the vibrios under the influence of the fluids of the body, greatly aids the study of the bactericidal substance. Before passing to the question of the part played by this substance in acquired immunity we must consider for a moment the principal properties of this acquired immunity. Very manifest in the peritoneal fluid, the power of causing Pfeiffer’s phenomenon is equally evident in the blood serum of immunised guinea-pigs, as has been demonstrated by Bordet. A drop of this serum, when quite fresh, readily and rapidly transforms a number of vibrios into granules. When the serum is kept for several days or has been heated to 55° C. for an hour, the total disappearance of the substance which produces Pfeiffer’s phenomenon is brought about. This at once betrays the presence of microcytase in the fluids of guinea-pigs that have acquired immunity against the cholera vibrio. Yet the blood serum and the peritoneal fluid of these animals, having been deprived of their microcytase by heating to 55° or 56° C., still retain a remarkable power over the vibrios. These organisms no longer undergo granular transformation, under the influence of the heated body fluids, but they are deprived of all power of motion, agglutinate into clumps and acquire a special susceptibility to the action of cytase. Soon after the discovery of Pfeiffer’s phenomenon, I[311] was able to demonstrate that this granular transformation can be obtained _in vitro_ under the following conditions. Prepare a hanging drop with the blood serum of a guinea-pig vaccinated against the cholera vibrio, a serum which has lost the power of transforming, by itself, the vibrios into granules. Add to it a drop of the peritoneal lymph of a normal unvaccinated guinea-pig; this lymph contains dead or living leucocytes and is, by itself, also incapable of producing Pfeiffer’s phenomenon. When, to the mixture of these two fluids, which are inactive when they are employed separately, a few cholera vibrios are added, they are quickly transformed into granules. This transformation, obtained _in vitro_, is remarkably like that produced in the peritoneal cavity of the vaccinated animal.

Jules Bordet[312], working in my laboratory, made a very complete investigation of Pfeiffer’s phenomenon outside the animal body and found that, in my experiment, the peritoneal lymph can be replaced by the blood serum of the vaccinated guinea-pig without thereby in the least hindering the granular transformation. After making a specially thorough study of the question he has come to the conclusion that Pfeiffer’s phenomenon is the result of the action of two substances. One of these is found in the blood serum and in the peritoneal fluid of guinea-pigs vaccinated against cholera, heated to 55°–56° C. or deprived by some other means of their individual power of transforming vibrios into granules. This substance resists this temperature and only loses its activity on being heated to 68°–70° C. The second of the two substances, that found in the peritoneal lymph or in the blood serum of the normal guinea-pig, is, on the other hand, destroyed at 55°–56° C. and is nothing but the ordinary cytase (or alexine) present in the fluids of normal animals.

[Sidenote: [227]]

The facts we have described with regard to Pfeiffer’s phenomenon in the body fluids of immunised animals must, then, be interpreted as follows. The fresh peritoneal exudation or blood serum of these animals readily produces the granular transformation, because in these fluids both the two necessary substances are found. But as microcytase is a very unstable substance which, under the influence of time or heating to 55°–56° C., is destroyed, the fluids of immunised animals are very readily deprived of it. The blood serum then, after being some time outside the body, becomes incapable of transforming vibrios into granules; but when to it is added a small quantity of the cytase, found in the blood serum or in the peritoneal lymph of the normal guinea-pig, the transformation takes place with great rapidity. To the serum of the immunised animal, which has become inactive, is restored its property of setting up Pfeiffer’s phenomenon. This interpretation, formulated by Bordet, corresponds to the whole of the known data and is now generally accepted.

[Sidenote: [228]]

As the fluids of immunised animals, that have become incapable of transforming vibrios into granules, still retain their power of rendering these organisms motionless and of uniting them into clumps, it might be asked whether this agglutinative substance might not be the substance, stable under heat, which is necessary for the production of Pfeiffer’s phenomenon. For some time, indeed, it was believed that this phenomenon is due to the microcytase acting on vibrios which have first been modified by the agglutinative substance. This latter substance resists heating to 55°–56° C., is only destroyed at higher temperatures, and is retained in the blood serum long after the cytase has entirely disappeared. The analogy between the agglutinative substance of the fluids of animals that have acquired immunity and the substance in the same fluids that is stable under heat is undeniable, and yet these two substances are not identical. A whole series of observations, which we shall presently describe, demonstrate this thesis clearly. A serum may be highly agglutinative without being capable of bringing about the transformation of vibrios into granules; the converse also holds good. The substance which sets up Pfeiffer’s phenomenon, and which is found in the fluids of immunised guinea-pigs, is a “fixative substance” analogous to those we have already met with in the serums of animals so adapted that they are able to resorb the various cell elements. As in the resorption of cells, so also in the destruction of micro-organisms, the fixatives are specific. The substance which aids the transformation into granules is not only distinct from the fixatives which sensibilise red blood corpuscles or spermatozoa, but also from the fixatives which sensibilise bacteria. This specificity has been demonstrated and carefully studied by Pfeiffer, who has shown that it may even serve to distinguish species of bacteria. The serum of a guinea-pig which has been immunised against the cholera vibrio, will render sensitive these vibrios, and these only, to the action of the microcytase. Even allied vibrios, such as various water vibrios, for example, are not sensitive to the fixative of anticholera serum. On the other hand, the serums obtained after the inoculation of these aquatic vibrios are incapable of producing granular transformation in the cholera vibrio.

When we inject into one and the same animal several species of vibrios we obtain a serum or a peritoneal fluid which produces Pfeiffer’s phenomenon with the vibrios of all the species which have been used to make the inoculations. This antivibrio serum contains only a single cytase for the vibrios, but contains as many different fixatives as there were species inoculated.

[Sidenote: [229]]

The transformation of vibrios into granules, when produced in a high degree against virulent vibrios, under the influence of the body fluids of immunised animals, affords a very valuable indication of the simultaneous presence of cytase and of specific fixative. As we have already stated, at the commencement of this account of the acquired immunity of guinea-pigs against the cholera vibrio, Pfeiffer’s phenomenon is manifested in the peritoneal fluid of these animals in a very short time (5 to 20 minutes) after the inoculation of the vibrios. This proves that in this fluid the two substances really occur together, and that the fixative and the cytase are in solution in the plasma of the exudation. Is it the same in every part of the body of immunised guinea-pigs? If, instead of introducing the cholera vibrio into the peritoneal cavity, we inject it into the subcutaneous tissue or into the anterior chamber of the eye of these animals, Pfeiffer’s phenomenon does not make its appearance. The vibrios, isolated or collected into small clumps, do not undergo granular transformation; they keep their normal vibrio form and remain alive much longer than in the peritoneal cavity. Some of them may be found still living 24 hours after subcutaneous injection and several (4–6) days in the anterior chamber of the eye. Nor can Pfeiffer’s phenomenon be observed when the cholera vibrio is introduced into the oedema of the foot, produced in consequence of the slowing of the circulation, the vibrios remaining alive for a fairly long time. These facts clearly indicate that in the fluid thrown out in passive oedema, just as in the aqueous humour of the eye or in the subcutaneous tissue, the two substances necessary to set up the granular transformation are not present. Are both of them absent or only one? This question is easily answered on adding to the fluids mentioned normal guinea-pig’s serum, a serum which, by itself, is incapable of producing Pfeiffer’s phenomenon. Bordet[313] has made these experiments and found that when to the fluid of the passive oedema of the immunised guinea-pig normal serum is added, this fluid transforms the cholera vibrio into granules, but does so in less degree than does the serum of the same immunised guinea-pig, when heated to 55°–56° C., to which normal serum has likewise been added. There is, then, reason to conclude that the fluid of the oedema does not contain any cytase, but contains a certain quantity of cholera fixative, less, however, than that which is found in the blood serum. As to the aqueous humour of the eye of immunised animals, analogous experiments have demonstrated that it contains neither of the two substances necessary for the production of Pfeiffer’s phenomenon.

With the help of the facts I have here summarised, we arrive at the following conclusion. In the animal that is immunised against the cholera vibrio, microcytase is found in the peritoneal exudation; it does not pass, however, either into the fluid of the passive oedema or into the aqueous humour of the eye; the cholera fixative is found in the peritoneal fluid and passes into the oedema, but does not penetrate into the fluid of the eye. This indicates that microcytase is found in fluids rich in leucocytes, but is absent from those which contain very few or none of these cells.

The introduction of vibrios into the peritoneal cavity of immunised guinea-pigs at once produces Pfeiffer’s phenomenon, and at the same time causes the disappearance of the majority of the leucocytes from the peritoneal lymph. We have already had occasion, several times, to speak of this phagolysis, because it is produced as a sequel to the injection into the peritoneal cavity of blood, spermatic fluid, and all kinds of fluids. The greater the quantity of fluid injected and the greater the difference of the temperature between it and the contents of the normal peritoneum the more vigorous is phagolysis.

[Sidenote: [230]]

Pierallini[314], working in my laboratory, studying phagolysis in the peritoneal cavity of the guinea-pig, has obtained several results worthy of attention. Of all the fluids used by him, such as water, broth, filtered cultures of micro-organisms and physiological saline solution, the last of these caused the least intense phagolysis, yet one sufficiently well marked. Immediately after the injection of any of the above fluids the number of leucocytes in the peritoneal lymph diminishes very considerably, the cells being found collected in clumps on the omentum. Many of them exhibit signs of enfeeblement and of partial destruction. Alongside the leucocytes are found fibrinous masses, this affording evidence that some of the leucocytes have been greatly damaged and have given up the fibrin-ferment which induces coagulation of the fibrin. When Pierallini injected fluids containing coloured powders in suspension, such as Indian ink and vermilion, he observed that these substances accumulated on the greater omentum, which became stained black or red. Microscopical examination revealed the existence of a not very intense phagocytosis and a number of free coloured granules in the midst of filaments of fibrin.

[Sidenote: [231]]

The leucocytes which, during this phagolysis, allowed the fibrin-ferment to escape might also give up a certain amount of their microcytase. This microcytase would pass into the peritoneal fluid and give rise to Pfeiffer’s phenomenon. If this hypothesis be correct, the suppression of phagolysis would result in the absence of the transformation of the vibrios into granules. It is not a difficult matter to verify this hypothesis as we have a means of preventing phagolysis or at least of reducing it very considerably. Issaeff[315], in an investigation carried out in Pfeiffer’s laboratory, demonstrated that an intraperitoneal injection of physiological salt solution, broth, urine, etc., reinforces the leucocytes and brings them up in large numbers into the peritoneal cavity. It is easy to foresee that such an injection would serve to diminish the intensity of the phagolysis. In fact, if we first inject a few cubic centimetres of physiological salt solution or of fresh broth into the peritoneal cavity of a guinea-pig, and if, on the following day, we repeat the same operation, we shall find that after the second injection phagolysis is much less powerful than after the first. Pierallini, who repeated these experiments, observed that the phagocytosis of the coloured granules is much more complete in the guinea-pigs that were treated by a preliminary injection into the peritoneal cavity. The amount of fibrin on the omentum is in this case much less, and the phenomena as a whole show that in these guinea-pigs the damage to the leucocytes is very considerably attenuated.

We have been able to demonstrate that in the case where phagolysis is thus diminished, Pfeiffer’s phenomenon is not produced or is manifested in a very feeble degree. If the experiment succeeds, the fluid taken from the peritoneal cavity of a guinea-pig prepared the day before and then injected with a culture of cholera, is opaque and thick, like pus. It contains a mass of leucocytes in good condition, a large number of which gorge themselves in a few minutes with a number of vibrios. The plasma of this exudation contains few vibrios, and these retain their normal form and do not exhibit, save exceptionally, a granular change. A little later there remain no free vibrios; they are all contained within leucocytes. Pfeiffer[316] declared himself against the facts I have just summarised, because he was never able to prevent the granular transformation of the vibrios, in spite of the preparatory injection of sodium chloride. Abel[317], who repeated the experiments, expressed an intermediate view: in guinea-pigs prepared by injections the day before, he observed that one portion of the vibrios became transformed into granules, whilst another became the prey of the leucocytes. The fact is, the suppression of phagolysis demands special conditions: the broth that is injected must be freshly prepared, and before its introduction into the peritoneal cavity it must be heated to 37°–39° C. Even when these precautions are taken it sometimes happens that the experiment is not very successful. In making the experiment we must be guided by the appearance of the peritoneal fluid withdrawn into the small glass pipettes. If the fluid which enters the tube is clear or scarcely clouded, it indicates that phagolysis has taken place, in spite of the preparatory injection. The experiment is successful in those cases where the peritoneal exudation is very cloudy and resembles pus.

[Sidenote: [232]]

As the demonstration of the suppression of Pfeiffer’s phenomenon as well as that of phagolysis is of fundamental importance, I asked M. Garnier[318] to carry out further experiments with the object of setting the question at rest. Using a whole series of fluids for the preparatory injection he found that fresh broth gives the best results. In guinea-pigs in which the phagolysis had been reduced to a minimum, phagocytosis commenced immediately after the injection of the vibrios. In from two to five minutes many vibrios are found inside the leucocytes, the free vibrios now being few in number and not exhibiting Pfeiffer’s phenomenon. Garnier in his memoir gives photographic reproductions of leucocytes crammed with vibrios; these should convince the veriest sceptic. Since the publication of this paper no objection has been advanced, and this question of the suppression of the granular transformation of vibrios may now be regarded as definitely settled. I have since demonstrated this feature to many observers, all of whom have assured themselves of its accuracy. It must, then, be accepted that Pfeiffer’s phenomenon is not produced in the peritoneal cavity except when there is phagolysis. As this fact renders it very probable that the microcytase, which is necessary for the transformation of the vibrios, escapes from the injured leucocytes, it becomes necessary to verify this hypothesis by a series of other experiments. If this hypothesis be well founded, Pfeiffer’s phenomenon should not be observed in those situations in the body where there are no, or almost no, leucocytes already present. These conditions can be realised by injecting cholera vibrios into the subcutaneous tissue or into the anterior chamber of the eye of guinea-pigs that are well vaccinated against the cholera vibrio. Under these conditions, as I have demonstrated in my work on the extracellular destruction of cholera vibrios, the vibrios retain their normal form and are never transformed into granules. Pfeiffer has questioned this result, stating that beneath the skin of vaccinated guinea-pigs the granular transformation is always produced, though in a more feeble fashion and after more delay than in the peritoneal cavity. The contradiction between Pfeiffer’s experiments and my own can, however, be explained. When inoculating the vibrios into the subcutaneous tissue, or during the withdrawal of the exudation formed at the point of infection, small haemorrhages are sometimes produced and a certain amount of microcytase is set free from the leucocytes found in the effusion of blood; these cells also give up to the extravasated blood a portion of their fibrin-ferment. When the experiment is successful, that is to say when no haemorrhage is produced during the operations involved, the subcutaneous exudation contains normal vibrios only, without the appearance of any trace of Pfeiffer’s phenomenon in the fluid.

[Sidenote: [233]]

If the extracellular transformation of the vibrios into granules were the real cause of the acquired immunity, the absence of this phenomenon in the subcutaneous tissue of the vaccinated guinea-pig should lead to the death of the animal. As a matter of fact this does not take place and the animal resists the inoculation of the vibrios. This conclusion is open to one serious objection. As the cholera vibrio in the great majority of cases is incapable of producing a fatal infection when inoculated subcutaneously, even in normal unvaccinated guinea-pigs, this example of immunity must be placed in the category of natural immunity, a kind of immunity which may depend on causes other than those on which acquired immunity depends. To answer this objection it was necessary to select a race of vibrios capable, when injected subcutaneously, of causing death. Mesnil[319], chief of my laboratory staff, undertook to carry out experiments with the Massowah vibrio, which is regarded by some authors as belonging to the true cholera species. When inoculated subcutaneously into unprotected guinea-pigs, it induces local oedema, in which the vibrios swarm; the infection rapidly becomes generalised and causes the death of the animal in 24 hours. Yet this vibrio, when injected into the subcutaneous tissue of well vaccinated guinea-pigs, is completely resisted by these animals and not the least trace of Pfeiffer’s phenomenon is produced. Under these conditions, a certain number of the vibrios are at first united into masses, but a considerable number remain isolated and motile. Some hours after inoculation the number of clumps diminishes and the isolated vibrios become more numerous, a fact which indicates a certain amount of adaptation of the vibrio to the medium in which it now finds itself. But never, so long as the vibrios remain free in the subcutaneous exudation, do they become transformed into granules.

[Sidenote: [234]]

Salimbeni[320], in an investigation carried out in my laboratory, endeavoured to satisfy himself whether or no Pfeiffer’s phenomenon is produced in the subcutaneous tissue of a horse that had been hyperimmunised against the cholera vibrio. This animal had, during a period of 14 months, received considerable quantities of these microorganisms, and the serum of its blood transformed the vibrios into granules with great rapidity and intensity. In spite of such favourable conditions for the manifestation of Pfeiffer’s phenomenon, it was never produced beneath the skin of this horse. The vibrios when injected in this position became completely motionless in a very short time, but they kept their vibrio form and remained alive for a number of hours. The exudation drawn off up to 24 hours after inoculation still gave growths of the cholera vibrio.

As it is more easy to introduce, without effusion of blood, the cholera vibrio into the anterior chamber of the eye than beneath the skin, and as the aqueous humour contains no fixative, the absence of the granular transformation in the first of these two situations has been observed even by Pfeiffer himself. The demonstration of this fact presents no difficulty, and for a considerable period we may observe free and perfectly motile vibrios moving about in the aqueous humour. The exudation from the eye contains many of these living organisms, which when sown on culture media made their appearance as colonies even when the fluid has been withdrawn from the eye several days after inoculation.

[Sidenote: [235]]

These carefully established facts show very clearly that the microcytase is only met with in the fluids of the living animal in those situations in which there are many pre-existing leucocytes and under conditions in which the cells undergo a more or less marked phagolysis. This may be corroborated by a decisive experiment. When we inject a suspension of the cholera vibrio directly into the veins of a guinea-pig, well vaccinated against these organisms, and whose serum produces _in vitro_ Pfeiffer’s phenomenon with great rapidity, this phenomenon is not manifested. This experiment has been performed and described by Bordet[321]. Having injected a suspension of this vibrio into the jugular vein of a guinea-pig vaccinated against the cholera vibrio, he killed the animal an hour later and found, in the blood of the heart, vibrios that had kept intact their form and their property of staining with methylene blue. Cultivation of the blood of the heart, liver and spleen gave growths of vibrios. In another guinea-pig, hypervaccinated against the same organism and inoculated by the same method, the blood drawn off shortly (4–15 minutes) afterwards showed, in preparations treated with methylene blue, well-stained vibrios, of normal form and quite intact. This is the most direct proof of the absence of Pfeiffer’s phenomenon in the blood fluid of a living animal that enjoys a very marked acquired immunity. The intact vibrios were lodged inside the leucocytes.

Levaditi[322] repeated these experiments in my laboratory and varied the conditions under which the vibrios were injected into the blood vessels. He was sometimes able to observe phagolysis of the leucocytes of the blood and their almost complete disappearance from the peripheral circulation. In these cases the injured leucocytes accumulated in the pulmonary capillaries and masses of them were seen surrounding groups of vibrios that were transformed into granules. It was, however, easy to exclude phagolysis by preparing the animals by means of injections of physiological saline solution or broth. Under these conditions the leucocytes remained in the blood current and very soon ingested the vibrios. Whilst the vibrios that were still free in the blood plasma retained their form and staining power intact, those found inside microphages were already, in great part, transformed into granules. The rapidity with which these phagocytes ingest the vibrios and set up the changes in them is really extraordinary.

In this case, which affords a typical example of the reaction of the animal organism in acquired immunity, we see a very marked and immediate phagocytosis. It is this same process that has already been described as occurring in the peritoneal cavity of vaccinated guinea-pigs in which phagolysis was absent as the result of preparatory injection. In the subcutaneous tissue and in the anterior chamber of the eye, where Pfeiffer’s phenomenon is regularly absent, the phagocytosis follows its ordinary course and causes the destruction of the vibrios. This result has been confirmed repeatedly—see works by Bordet, Mesnil and Salimbeni already quoted.

[Sidenote: [236]]

We need only compare the extension of Pfeiffer’s phenomenon and that of phagocytosis in animals that are immunised against the cholera vibrio, to satisfy ourselves that the former phenomenon is a limited one whilst the latter is general. There might be advanced against the latter conclusion the fact of the absence of any ingestion of the vibrios in the peritoneal fluid of guinea-pigs that are immunised but are not preserved against phagolysis. When a little of the peritoneal fluid is drawn off with small tubes shortly after the injection of vibrios into the peritoneal cavity, as a matter of fact, only a very intense Pfeiffer’s phenomenon is seen, phagocytosis being completely or almost entirely absent. But this procedure is insufficient. If we are to get an idea of what really takes place in the abdominal cavity, the animal must be killed and the peritoneum and especially the omentum very carefully examined. As first demonstrated by Max Gruber[323] and later by Cantacuzène[324], the greater omentum is, in these cases, covered with a thick layer which contains a large number of leucocytes, of which some are filled with vibrios; further, this layer contains a mass of vibrios, in part transformed into granules, in part agglutinated or isolated and retaining their vibrionic form intact. As time goes on, the phagocytosis becomes more and more marked, and it is impossible to deny its existence or to attribute to it merely a secondary part.

We have seen that the suppression of Pfeiffer’s phenomenon in the peritoneal cavity and in the blood, or its total absence in the anterior chamber of the eye, does not in the least deprive the vaccinated guinea-pig of its acquired immunity. The animal resists the vibrios perfectly, without these requiring to be transformed into granules in the body fluids. This transformation does take place undoubtedly, but only inside the phagocytes. As already stated in the discussion on natural immunity (Chaps. VI, VII) the vibrios, after being ingested by the microphages, almost immediately undergo within these cells a change in form, very similar to that observed in the real Pfeiffer’s phenomenon. The microphages are often full of a quantity of granules, derived from the ingested vibrios, which in a short time are completely digested. This fact, of such constant occurrence in the phagocytosis of the vibrios, furnishes us with still another proof of the microphagic origin of microcytase.

[Sidenote: [237]]

If Pfeiffer’s phenomenon is merely a particular instance of the transformation of vibrios into granules in fluids containing microcytase, it is quite natural that its suppression should not involve a fatal infection of the vaccinated animals. On the other hand, if the phagocytic reaction, so widely different, really plays an important part in acquired immunity, everything that interferes with phagocytosis must at the same time compromise the refractory condition. With the object of solving this question Cantacuzène[325], working in my laboratory, undertook a detailed investigation of this point. He showed that the injection of opium, in a non-fatal dose, narcotised the guinea-pig and at the same time prevented the movements of the leucocytes. Small glass tubes containing cholera vibrios and introduced beneath the skin of vaccinated guinea-pigs, became filled with numbers of leucocytes in the non-narcotised animal; in the guinea-pig that had received tincture of opium, the tubes left for several hours contained no leucocytes and later only did they begin to enter the tubes. When a strong dose of cholera vibrios was injected into the peritoneal cavity of thoroughly vaccinated guinea-pigs, the animals easily resisted the inoculation. When, however, similarly vaccinated guinea-pigs were submitted to the influence of tincture of opium, the same dose of vibrios caused their death. In these narcotised animals, in spite of the considerable dilatation and hyperaemia of the vessels and in spite of the marked hyperleucocytosis of the blood, diapedesis was not produced during the first few hours after the injection of the opium, and it was not till later (5 to 6 hours after injection) that the leucocytes began to appear in the peritoneal cavity. The vibrios profit by the period of inactivity of the phagocytes to multiply, retaining their motility and also the property of staining with basic aniline dyes. When the retarded leucocytes make their appearance in the peritoneal cavity, they find it already invaded by a multitude of vibrios. In spite of this the leucocytes, especially the microphages, ingest an enormous number of the organisms; this does not prevent the death of the guinea-pigs, though it takes place some hours later than in the unvaccinated control animals. At the moment of death, free vibrios are no longer found in the exudation; they have all been ingested by the microphages, inside which they have undergone granular transformation. On making a post-mortem examination of the animal a large number of small heaps of vibrios, such as are never met with in animals that have not been submitted to the action of opium, are found on the omentum.

[Sidenote: [238]]

All that is necessary, then, is to retard the phagocytic reaction for a few hours in order to cause well-vaccinated guinea-pigs to succumb to the action of the vibrios. One can readily understand that, with this result before us, there can be no hesitation in attributing to phagocytosis a much more important part in assuring acquired immunity than to Pfeiffer’s phenomenon.

The study of other diseases produced by vibrios only serves to corroborate the general conclusions that follow from the detailed study of the essential processes in acquired immunity against the cholera vibrio. It is here necessary to recall the discovery by v. Behring and Nissen of the very marked bactericidal power of the blood serum of guinea-pigs that have been vaccinated against Gamaleia’s vibrio. When this fact was first demonstrated we were justified in thinking that the vibriocidal property of the blood might by itself explain this acquired immunity; but a comparative study of the phenomena which take place _in vitro_ with those which take place in the living animal, soon demonstrated how slight was the foundation for this hypothesis. Whilst the vibrios, when sown in the blood serum of hypervaccinated guinea-pigs, there perished in large quantities or even the whole of them, these same organisms, when inoculated into the subcutaneous tissue of the same animals, remained alive for several days. Gamaleia’s vibrio is much less capable of being transformed into granules than is the cholera vibrio, and we find it retaining its normal form even inside the leucocytes. There is no occasion in this case, therefore, to look for Pfeiffer’s phenomenon.

[Sidenote: [239]]

The rapid and marked destruction of Gamaleia’s vibrio, _in vitro_, in the blood serum of vaccinated guinea-pigs, and the prolonged survival of these organisms in the living animal, afford additional evidence that the two groups of phenomena cannot be identical. On the other hand, it furnishes a further proof that, during the preparation of the serum, there is produced, parallel with the coagulation, another process which confers bactericidal power on the serum. It is quite evident that, as in the case of the cholera vibrio, we have here to do with the liberation of microcytase at the expense of the destroyed or injured leucocytes. Acting along with the specific fixative of the body fluids, this cytase causes the death of the vibrios introduced into the serum. In the living organism, the microcytase not being free, these vibrios, although influenced by the fixative, resist until they have become the prey of the phagocytes. In an investigation which was the subject of a communication to the International Congress of Hygiene in London in 1891[326], I demonstrated that the phagocytic reaction is produced with great intensity in guinea-pigs that have been vaccinated against Gamaleia’s vibrio. The inoculation of this organism into the subcutaneous tissue, an inoculation which sets up a rapidly fatal infection in untreated guinea-pigs, gives rise in immunised animals to the formation of an abundant exudation, in which the numerous vibrios soon meet with resistance from the phagocytes. These phagocytes ingest the living vibrios, retaining them for some considerable time in their interior, but in the long run always digesting them completely. During the last phase of this struggle, we sometimes find, inside the leucocytes, vibrios that are transformed into spherical granules. It was with these cells, filled with ingested vibrios, that I was able first to carry out an experiment that has since been repeated again and again, always with the same result. When from a well-vaccinated guinea-pig a drop of subcutaneous exudation is withdrawn, at a stage when all the vibrios have for some time been ingested by the leucocytes, and transferred, in the form of a hanging drop, to the incubator at 35°–37° C., it is found that the ingested vibrios develop inside the phagocytes which have died outside the animal. The vibrios first fill the leucocyte and, continuing to multiply, cause the cell to burst when they distribute themselves in the fluid of the hanging drop (figs. 40 and 41). This experiment proves, in the first place, that the vibrios have been ingested alive, and, secondly, that the plasma of the exudation was incapable of preventing their later development.

[Illustration:

FIG. 40. Vibrios (_V. metchnikovi_) developed inside a microphage from a vaccinated guinea-pig. ]

[Illustration:

FIG. 41. Vibrios (_V. metchnikovi_) developed in a drop of exudation from a vaccinated guinea-pig. The vibrios have ruptured the microphage and scattered themselves in the fluid. ]

[Sidenote: [240]]

[Sidenote: [241]]

Having summarised the principal phenomena exhibited by vibrios in an animal possessing acquired immunity, we must now enquire whether the mode of destruction and disappearance taking place in these vibrios is of general application. Naturally, we commence this study with the spirilla, which in many respects present a great analogy to the vibrios. The task is an easy one, thanks to a very careful work recently published by Sawtchenko[327], on the _Spirochaete obermeyeri_ of recurrent fever. We know, from what has been said in Chapter VI, that the spirochaetes found in the serum of persons attacked by this organism, are, in the peritoneal cavity of guinea-pigs, destroyed by the intervention of the macrophages. These phagocytes guarantee the natural immunity of the guinea-pig against the parasite of recurrent fever. In guinea-pigs, into which blood or serum containing spirilla has been injected on several occasions, the destruction of these micro-organisms is effected in a different way. When Sawtchenko introduced a number of _Spirochaete obermeyeri_ into the peritoneal cavity of guinea-pigs so prepared, he noted that they underwent a transformation resembling that observed in Pfeiffer’s phenomenon. In a short time the majority of these micro-organisms assumed the form of very delicate spirilla to which were attached round granules. There was not a complete transformation of the spirilla into granules, but a portion of their contents exuded in the form of spherical drops. The spirilla that exhibited these changes lost their motility and collected into clumps. There was undoubtedly an extracellular transformation of the spirilla, but this took place only in the peritoneal cavity. When injected into the subcutaneous tissue of a prepared guinea-pig the spirilla brought about the formation of a firm but scanty exudation in this situation. In this exudation were found leucocytes containing spirochaetes which retained their normal form. These micro-organisms were found exclusively in macrophages and gave no evidence of the occurrence of Pfeiffer’s phenomenon. A like absence of this phenomenon was observed in normal guinea-pigs which had been injected subcutaneously with the same quantity of spirilla. In these animals, however, the oedema that appeared at the seat of inoculation was abundant and soft, and the disappearance of the spirilla, that is to say their ingestion by the macrophages, took place at a very much later period than in the prepared guinea-pigs. We have, therefore, in this respect a complete analogy with the vibrios: in both cases there is an absence of granular transformation below the skin and an ingestion by the leucocytes of the exudation; on the other hand, we have Pfeiffer’s phenomenon appearing in the peritoneal fluid. This analogy extends even further. Thus, in guinea-pigs prepared by repeated injections of human serum rich in spirilla, Sawtchenko could suppress Pfeiffer’s phenomenon in the peritoneal cavity just as easily as in the case of the vibrios. All he had to do was to inject a certain quantity of broth into the peritoneal cavity of his immunised guinea-pigs. Twenty-four hours later, on introducing spirilla into the animals at the same site, they retained their motility for hours, did not exhibit any granular transformation and were ultimately completely ingested by the macrophages.

[Sidenote: [242]]

These facts lead us to conclude that the fate of the spirochaetes of recurrent fever in the organism of guinea-pigs prepared by previous injections is governed by laws the same as those established for acquired immunity against vibrios. The spirilla are ingested and destroyed by the phagocytes, except where phagolysis occurs, in which case the cytase, being set free, attacks the micro-organisms outside the leucocytes.

[Sidenote: [243]]

After his discovery of the granular transformation of vibrios, R. Pfeiffer, in collaboration with several of his pupils, set himself to discover how far this phenomenon was general in acquired immunity. He directed his attention to the typhoid cocco-bacillus, upon which he had already published[328] a very detailed account of work carried out in conjunction with Kolle. These observers availed themselves of the discovery made by Beumer and Peiper[329], and Chantemesse and Widal[330] and confirmed by other observers, that laboratory animals, especially mice and guinea-pigs, could be easily vaccinated against the fatal disease set up by the micro-organism of typhoid fever. As in the experimental infection of the guinea-pig by the cholera vibrio, the vaccination of the animals against the typhoid bacillus could be carried out very easily, either by using sterilised cultures or the fluids of cultures deprived of their organisms by filtration. In the small laboratory animals a most marked acquired immunity may thus be obtained, and the study of the phenomena which appear in the vaccinated organism afforded evidence of a general analogy with those which have been observed when vibrios are used. In the peritoneal cavity of the immunised guinea-pigs, Pfeiffer’s phenomenon proper does not appear, that is to say, only a few of the bacilli are transformed into granules, the large majority retaining their bacillary form; still they are evidently greatly damaged: they become motionless and agglutinate more or less completely into clumps. If, however, a few of these micro-organisms are sown on nutritive media, they multiply freely and give abundant growths. The peritoneal fluid, then, acts most unmistakably upon the typhoid bacillus, but in a much less degree than does the peritoneal exudation of guinea-pigs upon the cholera vibrio when immunised against that organism. In both cases we have a pronounced phagolysis which sets free the microcytase, whose action on the vibrio is more marked than on the bacillus of typhoid fever. This extracellular action on the typhoid bacillus in the peritoneal cavity can be easily prevented by a previous injection, twenty-four hours before, of broth, physiological salt solution, or normal serum. The suppression of phagolysis is, as in the case of vibrios and spirilla, followed by the suppression of extracellular action on the typhoid bacilli.

The same analogy is observed in the phenomena which appear beneath the skin. The bacillus of typhoid fever, when introduced into the subcutaneous tissue of vaccinated guinea-pigs, although not appreciably injured by the fluid of the exudation, undergoes some agglutination. The injurious action of the fluids of the body is here still less effective than in the peritoneal cavity. But, as in the peritoneal cavity of vaccinated guinea-pigs previously treated with broth, so in the subcutaneous exudation it is the phagocytes which destroy the micro-organisms. In both cases there is a very great afflux of leucocytes, mainly microphages. These cells ingest and digest the bacilli, which ultimately disappear. The micro-organisms ingested by the microphages, once inside these phagocytes are transformed into granules very like those observed in the cholera vibrio similarly treated. In this respect the analogy between the two micro-organisms is complete.

[Sidenote: [244]]

Oppel, working in my laboratory, has repeated Cantacuzène’s work on the retarding action of opium upon the phagocytic process. He obtained the same results: under the influence of the narcotic, the leucocytes intervened only at a late stage, with the result that the vaccinated guinea-pigs succumbed to the typhoid infection. The same conclusion must be drawn from the experiments made by A. Wassermann[331]. Guinea-pigs that had been immunised against the bacillus of typhoid fever were completely resistant to a dose that was always fatal to the control animals. When, however, along with this dose of bacilli, a certain quantity (3 c.c.) of a serum which hinders the phagocytic reaction is injected, the guinea-pigs lose their immunity and die from typhoid infection. The serum employed by Wassermann was obtained from rabbits that had been treated with the blood serum of guinea-pigs. Rabbit’s serum, thus prepared, neutralises the action of the guinea-pig’s cytase, but, as demonstrated by Besredka[332], it also exercises several other functions, one especially, that of preventing phagocytosis. In Wassermann’s experiments it was the antiphagocytic function, then, that was the important factor in the suppression of the acquired immunity of the guinea-pigs. These experiments supply a fresh proof of the great importance of the phagocytic reaction in this kind of immunity, and afford further confirmation of the analogy between the mechanism of resistance of the animal’s organism against the typhoid bacillus and that against the cholera vibrio.

In presence of this striking analogy, it is unnecessary to insist further on the details of the acquired immunity of animals against the experimental disease set up by the micro-organism of typhoid fever. It will be better to select another example from the group of bacilli. Let us first take the acquired immunity against the bacillus of blue pus (_Bacillus pyocyaneus_) which for many years has been regarded as the best example in which to study this kind of immunity. Charrin, who was the first to obtain disease with this bacillus experimentally, published several notes[333] on the acquired immunity of the rabbit against it. He demonstrated the possibility of vaccinating this animal not only with living bacilli, but also with the products of their culture; he studied the blood serum of vaccinated animals, comparing it with the serum of normal rabbits, especially as to its action on the development of the _Bacillus pyocyaneus_. Although unable to find any bactericidal power properly so called in the serum of immunised rabbits, Charrin was the first to draw attention to certain modifications undergone by the bacilli when grown in this medium. He noted that under these conditions no pyocyanin was produced, and, in collaboration with Roger, he demonstrated that, in the serum of the vaccinated rabbit, the Bacillus pyocyaneus forms packets composed of little chains of greater or less length, whilst in the serum of the normal, susceptible rabbit, it develops in the form of normal rods, the rods for the most part being isolated.

[Sidenote: [245]]

From his experiments _in vitro_ Charrin concluded that there was marked enfeeblement of the functions of the _Bacillus pyocyaneus_ when submitted to the action of the vaccinated animal organism. Bouchard[334] has gone so far as to develop a theory of acquired immunity, in which the principal part is attributed to the impossibility of the micro-organism, after it has invaded the refractory animal, secreting its fluid products; there is no vascular dilatation and diapedesis does not take place. A comparative observation of the phenomena observed in rabbits that are susceptible to the pyocyanic disease and of those met with in vaccinated rabbits, most clearly, however, demonstrates the impossibility of accepting Bouchard’s interpretation. The inoculation of the bacillus of blue pus below the skin of the ear of the normal (unvaccinated) rabbit sets up extensive inflammatory reaction with marked hyperaemia; the diapedesis of the white corpuscles takes place at a comparatively late stage of the process and phagocytosis is neither set up nor completed until very late. On the other hand, in vaccinated rabbits, infected in the same way, the hyperaemia of the ear is insignificant, but diapedesis occurs very early and phagocytosis commences at once. It is not, therefore, the impossibility for the leucocytes to traverse the vessel wall, owing to the absence of the dilatation of the veins, which prevents them from making their way rapidly to the field of battle; it is their imperfect positive sensitiveness that is accountable for the tardy and incomplete phagocytosis. This interpretation is confirmed in other cases of acquired immunity.

[Sidenote: [246]]

More recently, Paul Müller[335] has laid special stress on the part played by the bactericidal action of the serum of animals that have been vaccinated against the pyocyanic disease. For him the negative results obtained by his predecessors lose their significance, since all their experiments were carried out under conditions of aërobiosis, whilst it is only in the absence of free oxygen that this bactericidal power can be exerted at all freely. Müller, therefore, set himself to compare under anaerobic conditions the bactericidal action on the _Bacillus pyocyaneus_ of serums coming from normal and from vaccinated animals. He succeeded in demonstrating that the blood serum of vaccinated animals is more bactericidal than that of normal rabbits. Before, however, drawing any conclusion from this observation, the following question must be answered: Are the phenomena observed _in vitro_ comparable with those seen in the living animal? In preceding chapters it has been shown so often that the blood serum obtained after the separation of the extravascular clot, can in no way be identified with the plasma of the circulating blood, that it is unnecessary to argue this matter further. If we wish to gain a clear idea of the mechanism of immunity in the living animal we must observe the course of events in the vaccinated animal and not draw conclusions from observations _in vitro_ except after strict examination. All the works on pyocyanic immunity above summarised lie under the reproach that in them this rule has not been adhered to.

Since the discovery of Pfeiffer’s phenomenon in animals that have been vaccinated against the cholera vibrio, much greater care has been taken to attend to the changes that occur in the animal that enjoys acquired immunity. Wassermann[336] was the first to attempt to apply Pfeiffer’s discovery to the _Bacillus pyocyaneus_. With a race of this bacillus rendered more virulent he succeeded in producing a fatal experimental malady in the guinea-pig against which he was able by various methods to vaccinate these animals.

[Sidenote: [247]]

He thus describes the phenomena observed in the peritoneal cavity of immunised guinea-pigs. Soon after injection the bacilli of blue pus become motionless, then “the rods swell up and melt, like wax in hot water. The formation of granules, such as occur in the cholera vibrio, has been observed but rarely. The process recalls rather that which takes place in experimental typhoid fever, as described by R. Pfeiffer. In all cases the phenomenon of solution takes place entirely in the fluid of the exudation, without any co-operation on the part of the leucocytes” (p. 284). We see that we have still to do with a kind of attenuated Pfeiffer’s phenomenon, without any granular change, but with an immobilisation of the bacilli. As Wassermann has remained satisfied with the examination of the peritoneal content which, as we know, gives but an imperfect picture of acquired immunity, Gheorghiewsky[337] set himself to study the question more thoroughly under my direction. With this object he vaccinated a series of guinea-pigs with living bacilli of blue pus, a sure method of obtaining acquired immunity. On examining the peritoneal fluid (withdrawn shortly after the injection of the bacilli) of the vaccinated guinea-pigs, he found that the bacilli were motionless and had undergone a certain degree of agglutination. They were not transformed into granules but became thicker and somewhat more dumpy. These changes are observed during the period of phagolysis, when only a few scattered leucocytes are to be found in the fluid of the peritoneal cavity. About two hours after the injection of the bacilli the leucocytes begin to reappear in the peritoneal exudation, more especially the microphages, which lose no time in seizing the bacilli, some of which become transformed into granules. A few hours later the exudation, containing a multitude of leucocytes, no longer contains any free bacilli: all are found inside the microphages. Nevertheless, if a drop of the exudation now be withdrawn and kept for some time at a temperature of 37° C., it will be found that the bacilli multiply inside the dead phagocytes outside the animal. We thus obtain colonies of bacilli, a fact which clearly proves that these bacilli whilst still alive have been ingested by the leucocytes. This experiment is, therefore, very similar to the one we have described in connection with Gamaleia’s vibrio.

[Sidenote: [248]]

Even at a later period, 24 or 30 hours after the injection of the bacilli, that is to say at a period when an examination of the exudation no longer reveals the presence of bacilli, the sowing of a drop of this exudation on a nutrient medium still gives isolated colonies of the _Bacillus pyocyaneus_ capable of producing the characteristic pigments. At a still later period, when the peritoneal exudation remains sterile, a post-mortem examination of the animals enables one to recognise, beneath the peritoneal surface, small white points made up of leucocytes. The sowing of these masses almost invariably gives colonies of the _Bacillus pyocyaneus_ which form blue pigments. We see from this account that, even in the peritoneal cavity of vaccinated animals, matters by no means go on in a uniform fashion, as would appear from Wassermann’s statements. Some bactericidal action in the peritoneal fluid there certainly is, but it is quite transient, and is limited to the period of phagolysis. The majority of the bacilli resist this attack of the body fluids to continue their struggle with the phagocytes, which, however, ultimately get the upper hand. In the subcutaneous tissue the part played by this phagocytic reaction is still more general. Gheorghiewsky has studied it not only in vaccinated guinea-pigs but also in a goat which had received several large injections of the _Bacillus pyocyaneus_. He observed that shortly after the subcutaneous injection of these bacilli, the fluid which accumulates at the seat of inoculation renders them motionless and in part agglutinates them. This fluid is clear and contains a few leucocytes and a number of bacilli which still retain their usual form. Some time later the leucocytes begin to come up to the seat of inoculation and to ingest the bacilli. At the end of 10 to 15 hours all the bacteria have been seized by the microphages and we no longer find any of them free. A hanging drop of this exudation, transported to the incubator, soon swarms with bacilli which have sprung from the organisms ingested by the leucocytes.

The exudation becomes more and more abundant at the seat of inoculation and ends in the formation of an abscess, from the contents of which cultures of the _Bacillus pyocyaneus_ may be obtained for a fortnight. The bacilli, however, finally disappear, this being due to the destructive action of the phagocytes and not to that of the fluid of the exudation.

This fundamental part played by phagocytosis in acquired immunity against the _Bacillus pyocyaneus_ has been confirmed by Gheorghiewsky by experiments on guinea-pigs vaccinated and then submitted to the action of opium. As in the analogous experiments of Cantacuzène on the cholera vibrio, the opium narcosis retards diapedesis and this, for some time, increases the chances of the bacilli. A tardy diapedesis and phagocytosis, no doubt, is produced which ends in the ingestion of the bacilli, but the animal loses its acquired immunity and finally succumbs in spite of the fact that the dose of _Bacillus pyocyaneus_ was insufficient to kill a control guinea-pig vaccinated to the same degree, but not submitted to the action of opium.

[Sidenote: [249]]

The example we have just analysed relates, then, to a micro-organism which is more resistant than are the vibrios, Obermeyer’s spirilla or even the typhoid bacillus, to the action of the microcytase which has escaped from the cells during phagolysis. The _Bacillus pyocyaneus_ undergoes, in the fluids of the vaccinated animal, the action of the specific fixative and can thus be rendered motionless and become agglutinated. But this action is insufficient to ensure immunity and should phagocytosis not take place in time to ingest the bacilli, the vaccinated animal succumbs. The reaction of the phagocytes is, therefore, indispensable if the acquired immunity is to be effective. In this respect the analogy is very great between the resistance of the vaccinated animal against the various bacteria (vibrios, spirochaetes, typhoid cocco-bacilli, bacilli of blue pus) that we have so far studied in this chapter. These bacteria have, however, this in common;—they are all endowed with a considerable power of motion. Pursuing our examination of the principal data on acquired immunity against micro-organisms, we must now choose examples from the group of non-motile bacilli; amongst these we assign the first place to the micro-organism of swine erysipelas. This small bacillus has been the subject of several important researches on acquired immunity, one of which at a certain period caused quite a sensation in the bacteriological world. Emmerich[338], in an investigation carried out in collaboration with di Mattei, made an unexpected announcement. He said he believed that he was justified in affirming that the acquired immunity of rabbits against the bacillus of swine erysipelas is due to the formation, in the fluids of the body, of an antiseptic substance which very quickly destroys this organism. This substance, secreted by the cells of the vaccinated animal, was supposed to act after the fashion of a solution of bichloride of mercury and to kill a large number of bacilli, introduced subcutaneously, in from 15 to 25 minutes. This discovery was not confirmed. In a series of experiments that I carried out[339] with the object of clearing up this question, and made under conditions as favourable as possible for the demonstration of the supposed bactericidal secretion, this action was never manifested. Not only did the virulent bacilli of swine erysipelas, when injected subcutaneously into well vaccinated rabbits, remain alive in the subcutaneous exudation for hours and even days, but the attenuated bacilli of Pasteur’s vaccines likewise remained intact. These bacilli when introduced into the anterior chamber of the eye survived for even a longer period. Here, as beneath the skin, the injection of the bacilli induced an exudation rich in leucocytes, amongst which microphages predominated. These phagocytes at once began to seize the bacilli which were destroyed not in the fluid of the exudation but within the leucocytes. Long after all the bacilli had been ingested, 24 hours and more after inoculation, the sowing of the exudation frequently gave growths in appropriate media.

[Sidenote: [250]]

Emmerich[340] sought by new experiments to remove these objections, but he found that the bacilli of swine erysipelas did not disappear from the vaccinated animal until some 8 or 10 hours after they had been introduced. There is, therefore, no longer any question of a rapid bactericidal action at all comparable to that of corrosive sublimate, which would destroy the introduced bacilli in less than an hour. The limit of 8 to 10 hours, accepted by Emmerich, is still too short and is not in accordance with my experiments; but even this was quite sufficient for the appearance of a free phagocytosis, a condition that really occurs. Emmerich has not directed his researches in this direction, and his theoretical conclusions did not in the least weaken the value of my arguments drawn from the demonstration of the ingestion and intracellular destruction of the bacilli by phagocytes.

[Sidenote: [251]]

The researches on immunity against swine erysipelas then languished for some time, until the discovery of Pfeiffer’s phenomenon gave a fresh stimulus to the study of this problem. One of Pfeiffer’s pupils, Voges[341], sought to apply the results obtained in the case of the cholera vibrio to the acquired immunity against the bacillus of swine erysipelas. He studied the blood serum of animals vaccinated against this bacillus and believed himself justified in affirming the existence of an acquired bactericidal power. Under no condition, however, did he observe anything comparable to Pfeiffer’s phenomenon, and he was compelled to admit that the bactericidal action of the serum is very feeble and only takes effect on young bacilli whose membranes are as yet very delicate and not very resistant. Mesnil[342] repeated these researches in my laboratory, but his results were very different from those obtained by Voges. The blood serum of rabbits, fully vaccinated against the bacillus of swine erysipelas, proved to be a good culture medium for this bacillus, and Mesnil affirms, as the result of numerous well-established observations, that “_in vitro_, the serum of rabbits immunised against the erysipelas has no bactericidal power or a very insignificant one.” On the other hand, the same fluid had a very marked agglutinative power. The bacillus of swine erysipelas, being non-motile, does not present the abrupt change that is observed in vibrios or in the typhoid bacillus when submitted to the influence of specific serums—under which conditions these organisms at once lose their motility. But the bacilli of swine erysipelas, when introduced into the specific serum of vaccinated animals, run together into masses which become more and more voluminous and fall to the bottom of the vessel, leaving a limpid supernatant fluid. When this bacillus is sown in the serum of vaccinated animals, it is seen to develop in the form of chains, composed of a large number of segments, which fall to the bottom of the tube. These bacilli, however, whether agglutinated or developed in chains, never show any attenuation in virulence. When the serum which bathes them is got rid of by washing, they are just as virulent as are the bacilli developed in the serum of normal unvaccinated rabbits. It is important to show that this virulence is kept up in spite of the fact that the bacilli, when placed in contact with the serum of immunised animals, become permeated with the specific fixative, as shown by the experiments of Bordet and Gengou[343]. These observers, indeed, have demonstrated that the bacilli of swine erysipelas, when kept for 24 hours in the specific serum heated to 55° C., acquire the property of absorbing the cytases contained in the unheated serum of normal animals.

The study of acquired immunity against the bacillus of swine erysipelas teaches us that this immunity is not due to any extracellular destruction comparable with Pfeiffer’s phenomenon; and that this immunity causes the production of a specific fixative and of a specific agglutinative substance, whose action on the resistance of the animal, to judge from the complete virulence of the bacilli when agglutinated and impregnated by fixative, is feeble or _nil_. It is the phagocytic reaction which is dominant in the immunised animals and which brings about the intracellular destruction of the bacilli.

The history of the anthrax bacillus, another representative of the group of non-motile bacilli, is particularly interesting, the more so that for some time the researches on acquired immunity have been concentrated almost entirely on the analysis of the facts observed in animals that have been vaccinated with the two Pasteur vaccines. In this way a large number of valuable facts have been collected; of these the more important may be presented to the reader.

[Sidenote: [252]]

In my first work on this subject[344] I called attention to the fact that in the rabbit vaccinated against anthrax, the bacilli, when inoculated subcutaneously, soon become the prey of leucocytes which accumulate at the spot menaced. In the unvaccinated control rabbits, however, the anthrax bacilli remain in a free state in the fluid of the subcutaneous exudation, only a few isolated rods being found inside phagocytes. I have since been able to confirm this fact[345], which must now be regarded as fully established. In the vaccinated rabbits the leucocytes exhibit a very marked positive chemiotaxis against the anthrax bacilli, whilst in normal unvaccinated rabbits the chemiotaxis of the leucocytes in the anthrax of the subcutaneous tissue is distinctly negative. When a small quantity of anthrax culture is inoculated subcutaneously into vaccinated and into unvaccinated rabbits there may be observed, even within a few hours, a very great difference. In the former there is found at the seat of inoculation an infiltration which swarms with leucocytes in the act of devouring bacilli. In the normal, susceptible rabbit, on the other hand, the exudation produced is soft, rich in fluid, and very poor in leucocytes. The vessels in the vicinity are distended with blood, and the fact that the leucocytes do not come up to the seat of inoculation is in no way due to the absence of vascular dilatation which might prevent diapedesis. The vessels are much more dilated than in the vaccinated rabbit, and yet in the latter the emigration is incomparably greater. This essential difference must be attributed to the sensitiveness of the leucocytes, which exhibit a negative chemiotaxis in the normal rabbit but a very marked positive chemiotaxis in the vaccinated rabbit.

It has been shown repeatedly that the subcutaneous exudation, very rich in leucocytes which have had time to ingest all the bacilli, when inoculated into guinea-pigs, ensures the appearance in them of a generalised and fatal anthrax; this affords evidence that the phagocytosis is exercised against virulent and therefore living bacilli. Marchoux[346], in Roux’s laboratory, has carried out numerous experiments on the vaccination of rabbits and has observed that the inoculated anthrax bacilli cause an exudation very rich in leucocytes, and that these cells ingest and destroy the bacilli. The phagocytes easily rid the refractory animal of the bacilli in the vegetative state, but the spores are much more resistant. After being devoured by the leucocytes they may remain inside them for months without germinating. Marchoux obtained cultures of anthrax from the subcutaneous exudation taken from vaccinated rabbits 70 days after inoculation.

[Sidenote: [253]]

The fact that the bactericidal action of the blood serum on anthrax bacilli is specially well marked in the rat, suggested the idea of trying to obtain, in this rodent, an augmentation of this property as a result of vaccination. Sawtchenko[347] attempted to do this in an investigation already cited in Chapter VI, carried out in my laboratory. He succeeded in thoroughly vaccinating white rats against virulent anthrax and in showing that the blood serum of these animals rendered refractory “is bactericidal in the same degree as that of non-immunised rats.” In the vaccinated rats “the subcutaneous exudation was as free from bactericidal substances as was the lymph of the control animals.” Sawtchenko was unable to demonstrate any increase of bactericidal power except in the peritoneal exudation of rats vaccinated by injection of cultures into the peritoneal cavity.

In spite, however, of the absence of any increase in the bactericidal property of the blood serum and of the subcutaneous exudation in vaccinated rats, the cell reaction obtained in them is very different from that met with in normal, susceptible rats. In a very short time (3 to 5 hours) after the subcutaneous injection of anthrax bacilli into the control rats (susceptible), an evident oedema is produced; in the vaccinated rat there is none. The exudation, not very abundant in the latter, already contains a number of leucocytes which are actively phagocytic, whilst in the control animal, examined simultaneously, “leucocytes are rarely met with, and few of them contain bacilli.” Later, the difference becomes still more marked. Pronounced oedema occurs in the control animal, it is poor in leucocytes but rich in bacilli, which continue to multiply; but “in the immunised rat, we find not a clear exudation but a thick and purulent fluid, full of leucocytes.” These cells devour all the bacilli; not a single one remains free. “Even after 14 hours bacilli ingested by the leucocytes are present and a culture of anthrax bacilli may be obtained from fluid taken from the seat of inoculation. Further, guinea-pigs or rats, when inoculated with a drop of this exudation (which contains no anthrax spores), succumb to anthrax.”

[Sidenote: [254]]

Even before these researches on the immunity of rats had been carried out, an attempt had been made to gain some idea of the differences presented by the vaccinated fluids of animals as compared with those presented by the fluids of control animals susceptible to anthrax. In 1886 I was able to demonstrate[348] that the anthrax bacillus develops abundantly in the defibrinated blood of sheep that had acquired immunity as the result of vaccination by Pasteur’s method. When these bacilli contain spores and are inoculated into rabbits they rapidly produce a fatal anthrax; but when no spores are present the injection of bacilli does not produce a fatal disease, and such infection is well supported by the rabbits. From this I concluded at that time that the anthrax bacillus must, in the blood of the vaccinated sheep, undergo a real attenuation in virulence, an interpretation which, as will be seen in the next chapter, was found to be erroneous.

Nuttall[349] showed that the defibrinated blood of refractory sheep acted as a nutrient medium for the anthrax bacillus. Making comparative investigations, by the plate method, on the bactericidal power of the blood of vaccinated and normal sheep, he observed that, in both cases, there was, at first, a certain decrease in the number of bacilli sown, more marked in the blood of the vaccinated than in that of the control animals. Nevertheless, 8 hours after the commencement of the experiment the anthrax bacteria had produced innumerable bacilli in the blood of the refractory sheep. Nuttall satisfied himself that this feeble bactericidal power was not to be compared with the very much greater power of the blood of the rabbit, an animal specially susceptible to anthrax.

More recently the properties of the serum of sheep which have been vaccinated against anthrax have been studied very carefully by Sobernheim[350]. He also was able to show that this serum allows of an abundant development of the bacillus, and that, outside the animal, it does not exercise any more appreciable bactericidal power than does the serum of the normal sheep. The serum of the best vaccinated sheep was found to be incapable of destroying even very small quantities of anthrax bacilli. The only change that Sobernheim could make out was with regard to the thickening of the bacterial membrane. This modification, however, was not constant and could not be seen in the serum of certain vaccinated sheep.

[Sidenote: [255]]

The serum of the sheep vaccinated by Sobernheim exhibited no increase of agglutinative power as regards virulent bacilli. Gengou[351], however, made it clear that repeated injections of cultures of the first vaccine of Pasteur into dogs produced a marked augmentation of this agglutinative power; but it was only produced when the attenuated bacillus was used. The virulent anthrax bacillus, developed as isolated rods, was not affected in the least by serum that was highly agglutinative for the bacillus of the first vaccine. Gengou also made the converse experiment with the serum of a dog into which he had previously injected a number of virulent anthrax bacilli. The dog, naturally refractory to anthrax, resisted the inoculation perfectly, but its serum did not acquire any agglutinative power against the first vaccine. He concluded therefrom that “the part played by agglutinins in the defence of the animal must be regarded as extremely problematical” (p. 339). On the other hand the phagocytic reaction in the vaccinated sheep is always very pronounced and constant. Von Behring[352], in one of his most recent publications, expresses the opinion that this example of acquired immunity must be placed in the category of phagocytic immunity.

In the group of bacilli, several examples of which we have studied, the typhoid bacillus approaches still more closely to the vibrios and spirilla in its relation to humoral properties. Here may be observed a kind of attenuated Pfeiffer’s phenomenon and somewhat profound modifications taking place under the influence of the serum of vaccinated animals. The _Bacillus pyocyaneus_ is more resistant to the injurious influence of fluids taken from immunised animals. This resistance is still more marked in the bacillus of swine erysipelas and again still greater in the anthrax bacillus. Whilst, however, these properties of the fluids of the body are found to be very variable and of unequal power, the phagocytic reaction is constantly manifested and always very actively. The leucocytes which, in susceptible animals, exhibit a very marked negative chemiotaxis or only a tardy and incomplete positive chemiotaxis, have, in the vaccinated animal, this positive susceptibility developed in a very high degree.

[Sidenote: [256]]

Before quitting the group of bacteria we must cast a glance at the mechanism of acquired immunity against representatives of the group of spherical micro-organisms. Amongst the cocci the streptococci have been especially studied as regards this immunity. For long great difficulties were encountered in vaccinating animals against these chain cocci, but Roger[353], Marmorek[354], Denys and Leclef[355] overcame these obstacles and succeeded in immunising the rabbit, one of the most susceptible species, to their pathogenic action. More recently the larger mammals, notably the horse, have been successfully immunised. A certain number of important facts, the knowledge of which is useful to complete the survey of the phenomena of acquired immunity, have thus been collected.

[Sidenote: [257]]

Roger set himself to study the properties of the blood serum of rabbits vaccinated against the streptococcus, and established the fact that this fluid had not the slightest appreciable bactericidal action; the streptococcus grew in it just as well as in the serum of fresh unvaccinated rabbits. When, however, he injected cultures grown in the serum of immunised animals into rabbits, these rabbits did not die and presented only transient and insignificant lesions. From this fact Roger concluded that there must be an attenuation of the streptococcus by the immune serum, a view which was shared by several other observers. In formulating this view, however, he had not taken into account the possibility that this serum acted not upon the coccus that had developed in it but upon the organism of the animal into which it was injected. Bordet[356], indeed, was able to show that the streptococcus which grows in the serum of immunised animals is in no way weakened in virulence. When he took a race very virulent for the rabbit (Marmorek’s streptococcus) and injected a minimal dose of a culture grown in the serum of immunised animals, the rabbits died just as did the control animals, because the amount of serum introduced was too small to exert any influence. So also, when he filtered this culture and got rid of the serum bathing the streptococci, it was found to be just as virulent as that grown in the serum of susceptible unvaccinated animals.

In confirmation of the discovery made by Roger with the serum of vaccinated rabbits, Bordet showed that the blood serum of horses highly immunised against the streptococcus did not exhibit any bactericidal action. Moreover, he found that this serum caused the development of somewhat agglutinated streptococci and that it was capable of throwing streptococci grown on the ordinary media into clumps. Summing up his researches on the properties of this serum Bordet concludes that it “causes no profound change in the streptococcus. The vegetative character of the coccus is not appreciably diminished, and its morphology remains the same save for certain variations in the length of the chains. Even the agglutinative power, recognised in numerous serums by recent researches, is, in the antistreptococcic serum, developed but slightly” (p. 196).

More recently von Lingelsheim[357] has studied the properties of the serum of animals which he had thoroughly vaccinated against the streptococcus. He observed a certain slowing of the development of the coccus in this serum as compared with the growth in cultures made in the serum of normal, susceptible animals. But this retardation was slight and transient, and exhibited itself especially in serums to which von Lingelsheim, following Denys, had added leucocytes.

[Sidenote: [258]]

Von Lingelsheim also noted a certain degree of agglutination of the streptococcus by the serum of vaccinated animals, although this was much more feeble than in the case of the cholera vibrio or the typhoid bacillus, when agglutinated by their corresponding serums. Speaking generally, he regarded the direct action of the body fluids as insufficient to bring about the rapid destruction of the streptococci in the vaccinated organism. “Since the action of the bactericidal substances is limited in time, the streptococci are able to adapt themselves to these substances and recover their former energy. As the phenomena of extracellular solution, of such a form as those observed under the influence of the cholera antibodies, are absent in the case of the streptococcus and as, on the other hand, a considerable ingestion of these organisms by the leucocytes is observed ... we must seek in the activity of these cells a second important element of the defence of the animal organism” (p. 78).

[Sidenote: [259]]

To Salimbeni[358], who has carried out in my laboratory an investigation on this subject, we are indebted for the most reliable information on the phagocytic reaction in acquired immunity against the streptococcus. He studied specially the phenomena in the subcutaneous tissue of a horse, hypervaccinated against Marmorek’s streptococcus; this animal received in all, at several injections, about five litres of living culture. In spite of this refractory condition, an oedema at the point of inoculation was soon produced; in this the micro-organisms remained free and the leucocytes were sparse. But the cellular reaction, at first insignificant, developed with great rapidity and many leucocytes, amongst which the macrophages were much the more numerous, were attracted. The phagocytosis was still delayed for some time, but it continued to increase and 20 to 24 hours after the inoculation it was complete. As soon as the phagocytosis was well established the oedema began to disappear. In the thick exudation, containing a mass of leucocytes, the macrophages are filled with a very large number of streptococci packed together. These cocci develop inside the cells, cause them to burst and again become free. A fresh arrival of leucocytes, however, takes place, this time mainly microphages. These microphages seize the free streptococci that have struggled so victoriously against the macrophages; this second phagocytic phase is final. The streptococci still remain alive inside the microphages for some days, but ultimately are killed and digested by the phagocytes. At a period when, 5 or 6 days after injection, insignificant or isolated traces of streptococci are to be found in the microphages, the exudation when sown in nutritive media still gives abundant cultures. The incidents of this struggle between the streptococcus and the animal organism demonstrate the important part played by the phagocytes. The fact that the macrophages perish and allow the cocci to escape, clearly proves that these cocci have been ingested alive and virulent, and consequently that the fluid of the exudation was incapable of destroying or even of attenuating them. The macrophages, also, were powerless to bring about this result and the intervention of the microphages was necessary to cause the disappearance of the cocci. It is, however, always the phagocytes which ensure the final resistance of the animal.

In presence of these very precise results obtained from the work of Salimbeni, a work which I followed very closely, the previous researches by Denys and Leclef (_l.c._) made under less favourable conditions on vaccinated rabbits are deprived of their importance. These observers wished to get an idea of the difference between the reactions of the animal organism (_a_) after the injection of streptococci into the pleural cavity of immunised rabbits, and (_b_) after injection into that of normal susceptible rabbits. They killed the inoculated animals and found a very marked diminution of micro-organisms in the pleuritic exudation of the former. This diminution could not be attributed to a lysis of the streptococci by the body fluids, because there were never any signs of such destruction. Nor could the phagocytosis, very feeble at first, be considered as the cause of the disappearance of a large number of the streptococci. Denys and Leclef put forward a third hypothesis, which attributed this disappearance to the rapid resorption by the lymph stream of the injected fluid containing the organisms. Going over the record of their experiments it will be seen that in vaccinated rabbits the quantity of pleuritic exudation was always very much less than in normal rabbits. In presence of this feature there is reason to ask whether, in the case of the streptococci, a large number of these organisms were not fixed, along with the leucocytes, on the walls of the pleura, as in guinea-pigs that are inoculated intraperitoneally? Instead of being satisfied with merely examining the fluid exudation, the surface of the pleura should have been scraped in order to ascertain whether the phagocytic reaction was localised in this region.

In any case such incomplete results on the active immunity of rabbits in no way weaken the positive results obtained in the subcutaneous tissue of the horse, in which the phagocytic reaction plays a really preponderant part.

This example of the streptococci completes our series of bacteria in which we have studied their relations with the properties of the animal organism that has acquired immunity. We have still to see whether the acquired immunity against micro-organisms of animal origin is subject to the same law as that against bacteria.

[Sidenote: [260]]

For some years past a zealous study of the infectious diseases produced by animal micro-organisms has been carried out. Besides malaria, which occupies a most important position, attention has been directed to certain diseases in domestic animals that are set up by endoglobular haematozoa and by flagellata, and a fairly large number of accurate data have been collected with regard to Texas fever and its parasite the _Piroplasma bigeminum_, as well as upon the epizootic diseases due to _Trypanosomata_ (Tsetse fly disease or Nagana, “Dourine,” etc.).

We are indebted to Smith and Kilborne[359] for the earliest information concerning the acquired immunity of Bovidae against Texas fever. R. Koch[360] then added some very precise observations on the immunity of calves which had been inoculated with parasites attenuated in the body of the tick (_Boophilus bovis_). Lignières[361], who devoted much attention to this question in the Argentine Republic, has discovered a sure method of vaccinating the Bovidae against the “Tristeza,” the local name for Texas fever. He brought to Alfort specimens of attenuated haematozoa, and in Nocard’s presence performed successful vaccination experiments. Lignières is now engaged in devising a practical method of ensuring immunity under the special conditions found in the home of the “Tristeza.” Up to the present, however, there are no sufficient data as to the mechanism of the acquired immunity in this case. We have fuller information as to the essential phenomena observed in the organism of the rat vaccinated against _Trypanosoma lewisi_. We owe to Mme. L. Rabinowitsch and Dr Kempner[362] the first important data as to the possibility of immunising white or piebald rats against the disease produced by the flagellated infusorian. They noted that these animals when inoculated with the blood of grey rats containing _Trypanosomata_ acquire a very transitory disease which, however, confers an immunity against any subsequent infection. The flagellated organisms disappear from the blood within a few weeks, after which fresh injections of these parasites have no pathogenic effect.

[Sidenote: [261]]

Laveran and Mesnil[363] confirmed these observations, and in addition made careful observations on the mechanism of this acquired immunity. After making several inoculations with blood containing _Trypanosomata_ into white rats, they made a study of the properties of the blood serum of these immunised animals. First they established the fact that this serum exerts no microbicidal action on the _Trypanosomata_, but it agglutinates them without, however, rendering them motionless:—“The masses may be resolved into rosettes in which the _Trypanosomata_, united merely by their posterior extremities, have their flagella free and motile at the periphery.”

Laveran and Mesnil then studied the phenomena evolved in the refractory organism. When injected into the peritoneal cavity of immunised rats the _Trypanosomata_ are not acted upon injuriously by the body fluids. They are, however, devoured by the leucocytes. Laveran and Mesnil thus express themselves on this subject: “... we have demonstrated clearly and repeatedly that the _Trypanosomata_ are ingested alive, perfectly isolated and very motile, by phagocytes, and we have followed the details of this process of ingestion which recalls that of the ingestion of spirilla by the leucocytes of the guinea-pig. We consider, therefore, that the immunity is phagocytic in character.”

[Sidenote: [262]]

The main facts on acquired immunity established in connection with the most diverse micro-organisms, facts just described, may already be said to lead to certain general conclusions. They indicate in the first place that acquired immunity is accompanied by phenomena more complicated than those observed in natural immunity. In the two categories of processes observed in acquired immunity the phagocytic reaction is the only one that can be said to be constant. We find it in those examples in which the influence of the fluids of the body is most manifest, as in the experimental cholera peritonitis of the guinea-pig, as well as in those cases where the humoral action is most feeble, as in anthrax or in the _Trypanosoma_ disease of rats. We have, however, still to establish the relations that exist between phagocytosis and the part played by the fluids of the immunised animal, in order that we may, as far as possible, present a general picture of the inner mechanism of acquired immunity against micro-organisms. To attain this result we must place the reader in possession of further well-established facts, and we must postpone its discussion to the following chapter, which will be entirely devoted to the above-mentioned problem.