Chapter VI: Natural Immunity Against Pathogenic Micro-Organisms (2)

As the dog, of all mammals, exhibits the greatest natural immunity from anthrax, it is very natural that in the bactericidal property of its blood the key to the enigma has been sought. Thus Nuttall[212] concludes from his experiments that the anthrax bacillus is readily destroyed by defibrinated dog’s blood. But, as this result was not in accord with my observations[213] that the bacillus is easily cultivated in dog’s blood, and as several observers, especially Lubarsch[214], had arrived at conclusions opposed to those of Nuttall, systematic researches were made for the purpose of solving this complicated problem. Denys and Kaisin[215] sought to remove the objections formulated against the explanation of the immunity of the dog as due to the bactericidal property of its blood by affirming that this power, which is absent in the inoculated dog, develops whilst the animal is under the influence of the bacillus. Immunity is reduced, then, in this case to the establishment of a new property in the fluids during the course of the struggle of the organism against the inoculated bacillus. None of the observers, however, who have repeated these experiments, _e.g._ Lubarsch[216] and Bail[217] were able to confirm the results of the Belgian observers. Denys himself, indeed, having resumed this study with Havet[218], had to reject the conclusions of his former work executed in collaboration with Kaisin. He is persuaded that their error was due to the fact that in their experiments _in vitro_, the living leucocytes ingested the bacilli and prevented their development. As the result of these new researches Denys and Havet have come to the conclusion “that the main, the predominating part of the bactericidal power of the dog’s blood must be ascribed to the leucocytes acting as phagocytic elements” (_loc. cit._ p. 15).

[Sidenote: [161]]

As a result of the investigations I have summarised the conclusion is forced upon us that the natural immunity of the dog from anthrax is a function of the phagocytes. In presence of this uniformity of the experimental results it becomes very important to make a more profound study of the phenomena that manifest themselves during the destruction of the bacilli by the phagocytes of the dog. What are the phagocytic elements which play the principal part in this struggle, and by what means do they attain this result? Gengou[219] undertook a detailed investigation in my laboratory to answer these questions. He was able to convince himself, in agreement with the statements of his predecessors, that not only was the serum of dog’s blood not bactericidal for the anthrax bacillus, but that the plasma of the blood is no more so. The fluid of the aseptic pleural exudation obtained after injection of gluten-casein, was likewise incapable of killing the anthrax bacillus. When Gengou, by means of centrifugalisation, isolated the leucocytes from these exudations, washed them in physiological salt solution, froze them, and then macerated them in broth, he obtained suspensions of white corpuscles, to which he added bacilli. He was able to demonstrate that when the exudations contained macrophages principally, as is observed in exudations taken at the end of two or three days, the bactericidal power of the suspensions was _nil_ or insignificant. When, on the other hand, the leucocytes came from exudations only twenty-four hours old and were composed almost exclusively of microphages, the destructive action on the bacilli of the extract of the microphages in broth was most marked. Now it is fully demonstrated that in the exudation set up in the refractory dog by the injection of anthrax bacilli, it is especially the microphages which exhibit the phagocytic reaction against this bacillus.

This is how the question of the immunity of the dog from anthrax stands at present. The natural immunity of this species, which although not unlimited, is very real, depends on the activity of phagocytes. These elements, under the stimulus of the bacillus and its products, exhibit a positive chemiotaxis of the most marked character, they approach the bacilli, ingest them by a physiological act, and destroy them by means of a substance which is not found in either the plasma or the blood serum, but which can be demonstrated in an extract of the microphages.

[Sidenote: [162]]

In spite of the uniformity and precision of these data, it is impossible to rest satisfied with describing, as an example of natural immunity from anthrax, the single case of the dog. If the resistance of the rat against this disease was merely of historical interest because of the large number of works devoted to this question, we might relegate it to the chapter reserved for the history of our knowledge on immunity. But it is not so. The anthrax of rats is a subject full of very valuable instruction, and von Behring was quite justified in saying that whoever wished to get a true conception of natural immunity from a virus should pay special attention to this example.

As a matter of fact, it may be stated that the grey rat (_Mus decumanus_), the black rat (_Mus rattus_), and white rats are far from enjoying a true immunity from anthrax. They, nevertheless, exhibit a more or less marked resistance against this disease and are always less susceptible than are the other laboratory rodents: mice, guinea-pigs and rabbits. Rats resist attenuated bacilli (anthrax vaccines) better than do these three species, and in order to induce in them fatal anthrax it is necessary to inoculate a much larger number of virulent bacilli. On the other hand, rats are distinguished by a great irregularity in the resistance they offer to the bacillus. At times they resist very virulent bacilli; at others they contract a fatal disease after an injection of very attenuated bacilli (Pasteur’s first vaccine).

[Sidenote: [163]]

In my first memoir on anthrax[220] I noted the fact that in rats the phagocytosis against the bacillus when injected subcutaneously was more marked than after the same inoculation into the rabbit and guinea-pig. Later, this fact was disputed by several observers, who refused to accept the extent and importance of the phagocytic reaction in the rat. This opposition was strengthened by a very interesting discovery made by von Behring[221], namely, that the blood serum of the rat possessed a remarkably destructive power for the anthrax bacillus. When this observer added a certain quantity of anthrax bacilli to some blood serum of the rat, instead of elongating into filaments and dividing they underwent a change, lost their normal refraction and took on staining reagents very imperfectly. The membranes alone remained of the bacilli thus treated. Von Behring considered that this bactericidal action of the serum depends on the presence of an organic base dissolved in the blood fluid. He had merely to neutralise the serum by means of an acid, and there was at once a very abundant development of the bacillus. From these researches von Behring came to the conclusion that the natural immunity of the rat from anthrax can be reduced to terms of the chemical action of the blood on the bacillus.

In one of his most recent publications this author[222] returns to the question of anthrax in rats and sums up his present point of view as follows. He regards the immunity of these rodents as being relative, not absolute. “The anthrax bacilli”—he says—“die in rat’s serum _in vitro_; and in the cases where the inoculation of these animals with the anthrax virus is not fatal, it is at least reasonable to assume that the blood fluid likewise produces this protection in the organism of the living rat. Now, an immunity that manifests itself without the aid of any activity of the cell must undoubtedly be regarded as being of a humoral character” (_loc. cit._ p. 202).

[Sidenote: [164]]

Let us begin by analysing the facts as presented in rats into whose subcutaneous tissue we have injected anthrax virus. A certain number of them resist, without exhibiting any lesion other than a certain exudative inflammation at the seat of inoculation. The exudation is, in this case, very rich in leucocytes which quickly exert their phagocytic function and destroy the ingested bacilli. In this reaction it is the microphages that play the chief part, the macrophages intervening later and in a much less pronounced fashion. Usually, however, the inoculated rats exhibit a more serious illness: the bacilli multiply at the point of inoculation and excite the formation of an extensive oedema, rich in serous fluid, transparent, and very poor in leucocytes. It is only later that these cells intervene in any considerable number. The exudation becomes thicker and turbid, the numerous white corpuscles devour the bacilli and cause their disappearance. Under the influence of this marked reaction the animals in most cases recover, as has already been established by Frank[223]. But even in those individuals which succumb to anthrax death occurs more or less tardily, an examination of the internal organs then revealing a considerable phagocytic reaction. The spleen, often of enormous size, contains numerous macrophages which are filled with normal or more or less altered bacilli. In the liver these macrophages, which have devoured several microphages and some bacteria, are also found (Figs. 22 and 23).

When instead of bacteria in the condition of rods, anthrax spores are inoculated subcutaneously or into the anterior chamber of the eye, we can observe their germination. There is developed a whole generation of bacilli which behave like those we have already described, that is to say, they excite an exudation and are ultimately digested within the phagocytes (Figs. 24 and 25). All these phenomena of phagocytosis I described in detail more than ten years ago in my memoir on the anthrax of rats[224]. Since then not a single fact has been brought forward to invalidate the results there set forth.

FIG. 22. Macrophage from the liver of a rat affected with anthrax.
]

FIG. 23. Macrophage containing bacilli, from the liver of a rat
affected with anthrax.
]

FIG. 24. Microphage of rat filled with bacilli.
]

FIG. 25. Two microphages of rat that have ingested bacilli.
]

[Sidenote: [165]]

How is this paradoxical fact to be explained, that anthrax which grows in the body of the rat, there setting up a disease more or less grave and sometimes fatal, is so readily destroyed by the serum and blood when removed from the organism? From numerous experiments, carried out by Hankin[225] and by Roux and myself[226], it has been demonstrated that the bactericidal power of the fluids of the rat cannot be invoked as the cause of the animal’s resistance to anthrax. Those rats which show themselves very susceptible to this disease and die from anthrax infection, furnish, nevertheless, a serum that will prevent anthrax in other rats, and which will protect even mice into which the bacilli have been injected. Rats into which we inoculate on one side of the body a little anthrax culture, and on the other side the same quantity of bacilli mixed with blood serum from the same animal, manifest oedema at the former place only. It is from this latter point that the general infection takes place, the side where the anthrax bacilli mixed with serum was introduced remaining unaffected. Sawtchenko[227], who has investigated the immunity of the rat in my laboratory, has to the facts just mentioned added the observation that when the injection of bacilli causes haemorrhage the rat survives. When, on the contrary, the injection is made with a fine needle and without effusion of blood, the rat contracts a fatal anthrax.

[Sidenote: [166]]

[Sidenote: [167]]

It follows from these facts that the blood, immediately it has escaped from the vessels, undergoes a change in its composition and becomes bactericidal for the anthrax bacillus, whilst, when it is circulating in the organism, it exhibits no such power. Sawtchenko has studied the substance in the serum which kills the bacilli and has demonstrated that it will resist heating to 56° C.; even when heated to 61° C. the serum still exercises a certain amount of bactericidal power for very attenuated bacilli (Pasteur’s first vaccine). Researches on the distribution of this bactericidal power in the living rat have convinced Sawtchenko that none of it passes into the fluid of the passive oedema set up by the slowing of the circulation, nor into that of the active oedema developed as the result of the inoculation of anthrax bacilli. He observed that even the bacillus of Pasteur’s first vaccine grows abundantly in the oedematous fluid produced by the injection of virulent bacilli. The peritoneal lymph, however, exerts a very marked bactericidal action on the bacilli. Having demonstrated this fact Sawtchenko put to himself the question: May not the great difference between the action of these fluids depend on the fact that the lymph is rich in leucocytes, whilst in the fluid of the oedema they are almost absent? Pursuing this question, Sawtchenko made a comparative study of the bactericidal power of the serum, prepared outside the body, and of the blood plasma obtained by means of an extract of the heads of leeches, and he concluded from his researches that the bactericidal substance circulates in the plasma of the living rat and that it is not derived from the microphages, but must be looked upon rather as a secretion of the macrophages in the blood and of endothelial cells. This result was not confirmed by Gengou[228], who also took up the study of this important question in my laboratory. Instead of preparing the plasma by means of the addition of an extract of leeches he made use of a method much more perfect and free from sources of error. He introduced no foreign substance capable of affecting the results of his experiments. Collecting the rat’s blood in paraffined tubes, and centrifugalising it in similar tubes, he obtained a fluid which approaches much more closely the plasma of circulating blood than does serum. This fluid, however, will coagulate at the end of a fairly long interval, which proves that it cannot be looked upon as blood plasma. Gengou examined the bactericidal power of the fluid portion of the “plasma,” obtained by the process just described, on the anthrax bacillus, and also that of serum prepared in tubes in the ordinary way. The difference between the two fluids is very marked; whilst the serum destroys the bacilli sown in it very rapidly and dissolves their contents, the fluid of the “plasma” has no similar action. These results, confirmed several times, demonstrate very definitely that the plasma of the circulating blood does not contain any bactericidal substance. This, during the life of the animal, is found inside leucocytes and only escapes from them when the cells burst or undergo profound lesions, this taking place when the clot is formed and when the serum is prepared outside the organism, or in the effused and coagulated blood, or again in the peritoneal lymph during phagolysis. This phagolysis is inevitably produced as a result of rapid injection of foreign fluids into the peritoneal cavity, _e.g._ of broth or of physiological salt solution, containing bacteria in suspension.

[Sidenote: [168]]

The facts we have brought together on the subject of anthrax in rats form a whole whose several parts are in complete harmony. The phagocytes of this species of rodent contain a bactericidal ferment, a kind of cytase, which resists temperatures approaching 60° C. This cytase is very active against the bacilli, but in the living animal it can only act within the phagocytes, or, in a transitory and incomplete fashion, outside these cells, when phagolysis is taking place in the peritoneal cavity. The resistance offered by the rat to anthrax depends, then, on this phagocytic activity. For its manifestation it is necessary, first of all, that the phagocytes should manifest a positive chemiotaxis for the bacilli, and then that they should seize and ingest these organisms. These are the vital acts that decide the result of the struggle. When the phagocytes show themselves inactive the bacilli multiply in the oedematous fluid which contains no bactericidal cytase, and pass into the plasmas of the lymph and of the blood, which also are incapable of killing these bacteria. The animal may, then, die of anthrax, in spite of the presence in its body of a large quantity of bactericidal cytase which is to be found in situations to which the bacilli have not penetrated. In those cases, on the other hand, where the phagocytes accomplish their function, where they rush up to the menaced point and devour the inoculated bacteria, these bacilli are placed in contact with the intracellular cytase and undergo complete digestion. The organism in this case gets rid of its enemies and victoriously resists infection.

[Sidenote: [169]]

Anthrax in rats, then, presents one of the most instructive examples of natural immunity. But the detailed analysis of the mechanism of this resistance demonstrates very clearly the great part played by the phagocytes in this process. In this respect the organism of the rat presents, in a general fashion, a great analogy to the natural immunity of the dog, of birds, and of other representatives of the animal kingdom that we have examined. Under these conditions it is useless to insist at any length on other examples of resistance against anthrax which, moreover, have relation much more often to a natural immunity against attenuated bacilli than to one against true anthrax virus. Rabbits and guinea-pigs, so sensitive to this virus, often resist the inoculation of Pasteur’s vaccines. The rabbit is, in general, refractory to the first anthrax vaccine; it may even resist the second vaccine. The guinea-pig, a more sensitive animal, does not exhibit any natural immunity except against the first vaccine. In all these cases the mechanism is similar to that which the rat and the dog oppose to virulent anthrax. The bacilli, into whatever part of the body they are injected, set up an exudative inflammation which brings up a large number of leucocytes to the point menaced. These cells readily exert their phagocytic function and rid the organism of the introduced bacteria. In order to obtain a complete grasp of the part played by this reaction it will be found useful to inject beneath the skin of one ear of a rabbit a little anthrax vaccine and beneath the skin of the other the same quantity of virulent bacilli. The difference between the reaction in the two cases is very striking. The ear inoculated with vaccine soon becomes the seat of a circumscribed inflammation with a purulent exudation, all the bacilli in which have been devoured by the leucocytes. The other ear, on the contrary, presents, around the injected virus, only a serous or blood-tinged exudation containing no, or few, leucocytes; the bacilli are found free in the liquid and multiply without let or hindrance. Meeting with no opposition the virus becomes generalised throughout the organism and brings on death by anthrax septicaemia. Rabbits, into which anthrax vaccines only are introduced, oppose to the invasion of the bacilli a leucocytic barrier which arrests their extension. The natural immunity of the sheep, rabbit and guinea-pig is also a phagocytic immunity, but it is only capable of being exercised against bacilli previously attenuated in virulence. The researches of Mme Metchnikoff[229] on the reaction of the phagocytes of these animals to the bacilli of Pasteur’s two anthrax vaccines have demonstrated the importance of the destruction of these bacilli by the leucocytes. All the other examples of natural immunity against anthrax are also merely relative. The fowl that resists an anthrax virus strong enough to kill an ox or a horse, succumbs to a special variety of anthrax cultivated by Levin[230]. The dog, as we have seen, in spite of its pronounced natural immunity against anthrax, is killed by the special anthrax bacillus prepared by Martel.

In this immunity against anthrax we have to deal with a bacillus capable of living and reproducing itself in extremely varied media. Hence the reason, it may be said, that the bactericidal influence of the fluids is so little pronounced in this case. To bring it into relief we must, therefore, choose a bacterium less capable of adapting itself to the chemical composition of various culture media. In this matter we cannot do better than select pathogenic spirilla of extremely delicate nature and analyse the mechanism of the natural immunity of certain species of animals with respect to them. It must not be forgotten, however, that here we are making use of representatives of an infinitely small minority of pathogenic bacteria, the majority resembling the anthrax bacillus in the facility with which they can be cultivated in all sorts of nutritive media.

[Sidenote: [170]]

[Sidenote: [171]]

FIG. 26.—Leucocyte of guinea-pig in the act of ingesting two spirilla.
]

FIG. 27.—The same leucocyte, half-an-hour later.
]

FIG. 28.—The same leucocyte, ten minutes later than Fig. 27.
]

FIG. 29.—Leucocyte of guinea-pig in the act of ingesting a very active
spirillum.
]

FIG. 30.—The same leucocyte, forty minutes later.
]

FIG. 31.—The same leucocyte, half an hour later than Fig. 30.
]

The spirillum of recurrent fever of man (_Spirochaete obermeyeri_) was the first pathogenic microbe found in an infective disease distinctly human. Discovered a third of a century ago, it has passed through the hands of the most skilful bacteriologists, who have tried all possible methods of cultivating it outside the body. Koch himself tried to solve the problem, but, in spite of his incomparable skill, did not succeed. Later, Sakharoff[231], at Tiflis, discovered a spirillum very similar in appearance which produced a fatal septicaemia in the goose. He, also, tried to cultivate it, but in vain. His successors have not been more fortunate in this respect. Here, then, are two micro-organisms, against which natural immunity should be easily obtainable and in a fashion quite other than that against anthrax. Nothing, indeed, is more frequent than examples of very stable natural immunity against the spirilla of Obermeyer and of Sakharoff. As I wished to obtain a clear idea of the mechanism by which the guinea-pig resists injections of the spirillum of goose septicaemia (_Spirochaete anserina_) I made injections of goose’s blood, containing a quantity of these organisms, into the peritoneal cavity of guinea-pigs. This injection, as usual, causes the disappearance of most of the leucocytes, as the result of a very marked phagolysis. We know that, under these conditions, the damaged leucocytes allow a certain quantity of the bactericidal cytase to escape. In spite of this the spirilla remain intact and exhibit very active movements in the peritoneal exudation. This exudation, after a period of phagolysis, which lasts for two or three hours, begins to be stocked again with leucocytes which come up in increasing numbers, a fact that does not prevent the spirilla moving about with great rapidity. Even seven hours after the injection of goose’s blood we still find many extremely active spirilla among a large number of recently migrated leucocytes, some of which even at this stage contain red corpuscles of the blood of the goose. It is not until later that the ingestion of these spirilla by the leucocytes commences, the leucocytes at last damaging and completely destroying them. This act of phagocytosis may be readily observed in hanging drops of the peritoneal exudation of inoculated guinea-pigs. The attention of the observer is drawn to certain macrophage leucocytes which throw out one or two conical-looking processes (Figs. 26–28). These pseudopodia attach themselves to spirilla which exhibit very violent movements as though wishing to extricate themselves from the grasp of the leucocyte. Sometimes the spirillum succeeds in escaping, but usually it becomes surrounded by the protoplasm and sinks more and more deeply into the substance of the leucocyte. Even when almost surrounded the free part of the spirillum still continues to move (Figs. 29–31). These movements cease only after the complete ingestion of the spirillum. Once inside the phagocyte the spirillum is digested and soon becomes unrecognisable.

FIG. 32.—Macrophage of guinea-pig filled with spirilla of recurrent
fever (after Sawtchenko).
]

FIG. 33.—Macrophage of guinea-pig containing three _Spirochaete
obermeyeri_ (after Sawtchenko).
]

[Sidenote: [172]]

Recently, Sawtchenko[232] took advantage of an epidemic of recurrent fever at Kazan to make similar investigations on the natural immunity of the guinea-pig against Obermeyer’s spirillum. He observed that these organisms, when injected into the peritoneal cavity, remained there, alive, for 24 and even 30 hours, whilst these same spirilla, when kept at 37° C. outside the organism in their natural medium, died at the end of some (4–7) hours. The injection of human serum containing spirilla into the peritoneal cavity of guinea-pigs set up a phagolysis succeeded by a considerable afflux of leucocytes. In spite, however, of the arrival of quite an army of these cells, the spirilla continued to move rapidly; for a long time they evaded the phagocytes which, however, in the end always ingested them. But it is only the macrophages which fulfil their phagocytic function (Figs. 32 and 33); the microphages obstinately exhibit an absolutely negative chemiotaxis. Now, as the macrophages do not make their way into the peritoneal cavity until after the microphages have appeared, it is easy to understand that phagocytosis can only take place at a late period. Sawtchenko came to the conclusion that “in the peritoneal cavity of animals naturally refractory, the spirochaetes perish as the result of a slow phagocytosis and not from the action of the bactericidal substances of the fluids.” In conformity with this result this observer has often noted the ingestion of living spirilla by the macrophages, in hanging drops of the peritoneal exudation of inoculated guinea-pigs. The phenomenon corresponds exactly to that described in connection with the spirillum of the goose.

In spite of the great difference between the spirillum and the anthrax bacillus from the point of view of their adaptation to surrounding media, the general result is the same with both these microbes: animals endowed with natural immunity get rid of them through the agency of their phagocytes.

[Sidenote: [173]]

[Sidenote: [174]]

It would be impossible and even useless here to pass in review all the cases of natural immunity against infective micro-organisms. We must consequently limit ourselves to several examples which may have an interesting bearing on the study of the problem as a whole. The spirilla, whose history we have just recorded, remain in the peritoneal fluid, without change of form, up to the moment when they are captured by the macrophages. Let us see by what mechanism the natural immunity against micro-organisms, characterised by a very special sensitiveness to external influences and by a considerable change of shape, is produced. The cholera vibrio and its allies best satisfy this postulate. When they find themselves placed under unfavourable conditions, these vibrios immediately become transformed into small spherical bodies which are much more like cocci than vibrios. The cholera vibrio is pathogenic for the laboratory rodents, especially for the guinea-pig, when a fairly large quantity of a culture is injected into the peritoneal cavity. Against smaller doses, however, the natural immunity is a most marked one. If we take a race of the cholera vibrio of medium virulence, and inject into the peritoneal cavity of guinea-pigs a sublethal dose of a culture, the following phenomena may be observed[233]. The inoculated vibrios move actively in the peritoneal fluid, from which almost all the leucocytes have disappeared. There remain only a few lymphocytes which appear to be indifferent to the influences that set up a real phagolysis. But, little by little, fresh leucocytes come into the exudation and engage in a struggle with the vibrios which, so long as they are free, retain their curved form and complete motility. The microphages, especially, swarm into the peritoneal cavity. Some of them begin to ingest vibrios, but this phagocytosis is at first slight. Later it becomes much more active. The microphages and macrophages seize vibrios that are evidently living and uninjured, which, sometimes, may be observed inside the vacuoles of the leucocytic contents exhibiting very lively movements. Once ingested, however, many of the vibrios become transformed into round granules. This change of shape is constant when inside microphages, but is completely absent when inside macrophages (Figs. 34 and 35). Finally, the phagocytosis becomes complete, and the organism gets rid of the vibrios solely by means of this reaction. Even seven hours after injection of the vibrios, when the peritoneal fluid, crammed with leucocytes, has become thick and turbid, there still remain a few scattered vibrios which always retain their shape and their normal activity. A drop of this exudation, maintained at 38° C. outside the organism, gives, in a few hours, an abundant culture of very active vibrios. It must, therefore, be concluded that the fluid part of the exudation was powerless to destroy the vibrios or even to render them motionless, whilst the living leucocytes have shown themselves capable of ingesting and digesting them. The peritoneal exudation, withdrawn at a period when it no longer contains any free vibrios, still gives cultures of the organism for some time. Soon, however, there comes a period when the inoculated exudation remains sterile, this proving that the vibrios, ingested in a living state by the phagocytes, have at length been killed by the microphages and macrophages.

FIG. 34.—Microphage of guinea-pig filled with cholera vibrios, the
majority of which are transformed into granules.
]

FIG. 35.—Macrophage of guinea-pig filled with cholera vibrios not
transformed into granules.
]

[Sidenote: [175]]

When, instead of cholera vibrios of medium virulence, we take a variety completely deprived of pathogenic activity, it is sometimes observed that certain of these organisms, when injected into the peritoneal cavity of the normal guinea-pig, become transformed into spherical granules in the fluid of the exudation without any direct co-operation of the phagocytes. This transformation into granules was first studied by R. Pfeiffer[234] and hence has been termed Pfeiffer’s phenomenon. It is of limited occurrence in natural immunity and is produced, as I have been able to demonstrate, only under certain well defined conditions. Pfeiffer’s phenomenon is observed in the peritoneal fluid. It commences soon after the injection of the vibrios and takes place during the period of phagolysis. In other parts of the body of the guinea-pig, notably in the subcutaneous tissue and in the anterior chamber of the eye, Pfeiffer’s phenomenon does not manifest itself; the animal, none the less, resists the inoculation of the vibrios. Even in the peritoneal cavity, moreover, it is easy to check the granular transformation of the vibrios by means which prevent the production of phagolysis. When we inject into the peritoneal cavity of a guinea-pig a foreign fluid, capable of exciting the phagocytic action, e.g. veal broth, physiological salt solution, urine, etc., we first excite a transitory phagolysis. To this stage succeeds another in which the leucocytes become very numerous and much more resistant than before. If we take advantage of this period of leucocytic stimulation to inject vibrios which have been attenuated as much as possible, we shall observe that they soon become the prey of the peritoneal phagocytes, without manifesting any sign whatever of Pfeiffer’s phenomenon.

It is evident, then, that this extracellular destruction of the vibrios, sometimes observed in the peritoneal cavity, is really the work of the microcytase that has escaped from the phagocytes during their period of transient injury.

[Sidenote: [176]]

[Sidenote: [177]]

FIG. 36.—Peritoneal exudation from guinea-pig showing free
streptococci and microphages that have ingested _Proteus_ bacilli.
]

Having analysed the mechanism of natural immunity against certain bacilli, spirilla and vibrios, it will be interesting to determine whether the same rules are to be applied in the case of the cocci. Choice is not difficult since we may equally well fix upon the staphylococci, the pneumococci, streptococci or gonococci. Should we decide upon the streptococcus it is solely because the natural immunity against this micro-organism has attracted the special attention of several observers. A second advantage of the streptococcus, however, is the high degree of natural immunity manifested against it by a laboratory animal so convenient as the guinea-pig. Dr Jules Bordet[235] studied this subject in my laboratory. He observed that the injection of streptococci into the peritoneal cavity sets up a marked leucocytosis which ends in a complete destruction of the micro-organisms. The leucocytes rapidly ingest the great majority of the streptococci and destroy them; there remain only a few isolated and free individuals which are protected by a clear zone (aureola) which develops around them, but in the end they also become the victims of the voracity of the phagocytes. When we increase the dose of streptococci injected, phagocytosis still goes on, but some of the streptococci succeed in escaping, and we see a new generation produced which is distinguished by the thickness of the protective aureola. In spite of the afflux of a large number of leucocytes, they no longer ingest the streptococci and generalisation of the infection results, followed by the death of the animal. Natural immunity, then, can be suppressed under certain definite conditions. Dr Jules Bordet[236] wished to satisfy himself whether the leucocytes failed to fulfil their phagocytic function because of the paralysis of their movements, or as the result of some other weakness. With this object he injected into the peritoneal cavity of guinea-pigs, at the moment when the streptococci begin to get the upper hand of the leucocytes, a definite quantity of a culture of _Proteus vulgaris_. These small bacilli in a short time become the prey of phagocytes which, however, still refuse to ingest streptococci (fig. 36). There is thus in the peritoneal cavity a kind of selective process as regards the ingestion of these microbes. The _Proteus_ disappears as the result of phagocytosis, whilst the streptococci thrive in the fluid of the exudation and continue to multiply. This experiment, which readily succeeds, demonstrates very clearly the difference between the positive susceptibility of the leucocytes (with respect to the _Proteus_) and the negative (with respect to the streptococcus). Bordet, in accordance with the view now generally accepted, regards this sensitiveness as a chemiotaxis, that is to say a perception of the chemical composition of the surrounding medium. It must be admitted that the substance which excites the chemiotaxis of the leucocytes does not readily diffuse and may not, therefore, be found in a state of solution in the plasma of the peritoneal exudation. Otherwise the leucocytes would refuse to ingest, not only the streptococci, but also the small _Proteus_ bacilli, bathed in the same repellent fluid. It is more probable that the substance which excites the negative chemiotaxis is contained in the aureola that surrounds the streptococci, from which it only escapes with difficulty and for a short distance.

[Sidenote: [178]]

Marchand[237] continued the investigation of the same subject in Denys’ laboratory at Louvain. He studied the natural resistance of the guinea-pig, rabbit and dog against the streptococcus. He, also, came to the conclusion that phagocytosis constitutes the principal means of defence of these mammals in their struggle against one of the most formidable of the pathogenic micro-organisms. Starting from a single colony, Marchand obtained two distinct races, one very virulent for the rabbit, the other encountering a most effective natural resistance. This resistance is due to the activity of the phagocytes which destroy the streptococci in the ordinary fashion. He states as the general result of his investigation that “an attenuated streptococcus is a streptococcus readily devoured by phagocytes” whilst “a very virulent streptococcus is a microbe that is not attacked by the leucocytes,” and he adds that “a streptococcus is virulent because it is not devoured by phagocytes” (_l.c._ p. 270). Up to this point the views of Marchand are in accord with those of Bordet; but here they diverge, in fact as soon as it becomes a question of the explanation of the origin of the difference in the behaviour of the leucocytes. Marchand refuses to apply the theory of chemiotaxis and asserts “that the phagocytosis depends on some physical property of the streptococcus and is consequently dependent on the tactile functions of the leucocytes” (p. 292). The experiments upon which he founds his conclusion cannot, however, be regarded as absolutely demonstrative. Thus, Marchand observed that the attenuated streptococci, when conveyed in the culture-fluid of the virulent variety, are as readily devoured by the phagocytes as when they were injected alone. According to him, therefore, there was in the culture-fluid of the virulent streptococcus no soluble substance capable of exciting the negative chemiotaxis of the leucocytes. But is it quite proved that this substance must necessarily pass into the filtrate of a virulent culture? If it adheres closely to the glairy aureola, as we have suggested, may it not remain behind with the bodies of the streptococci, without passing through the filter in any appreciable amount? The question cannot be regarded as definitely settled, but probability appears to be on the side of the theory of chemiotaxis.

Marchand also investigated whether the immunity against the attenuated streptococcus might not be explained by the bactericidal activity of the fluids of refractory animals. His results were unvarying and definite. The blood serum of his animals never exhibited any bactericidal power against the streptococcus, and the attenuated race, like the virulent one, grew well in the serums of the rabbit, dog and guinea-pig.

More recently, Wallgren[238] has taken up the study of the immunity and susceptibility of rabbits with respect to the streptococcus. His conclusions are, on the whole, in accord with those of his predecessors. He found that if the injected streptococci were not very virulent phagocytosis began immediately after the injection into the peritoneal cavity and continued as long as there were any streptococci to be attacked. In those cases, on the other hand, where the streptococcus was endowed with a greater virulence, a transitory phagocytosis took place at the beginning of the infection; but the streptococci soon succeeded in adapting themselves to the struggle with the leucocytes and kept them at a distance. The multiplication of the streptococci could then go on without restraint and the animal soon succumbed to a generalised infection. Wallgren considers that, in the defence of the organism against the streptococcus, the products of the destroyed leucocytes may, sometimes, play a part.

[Sidenote: [179]]

As the mechanism of natural immunity against the groups of bacteria—bacilli, spirilla (and vibrios) and cocci—presents a very great analogy in all three, it might be considered superfluous to continue our analysis of this phenomenon. Our review, however, would be incomplete if we omitted to take note of the natural immunity of the animal organism against micro-organisms which are distinguished by an exceptional toxicity. The first place in this group must undoubtedly be assigned to the bacillus of tetanus.

FIG. 37.—Leucocytes of rabbits filled with tetanus spores.
]

[Sidenote: [180]]

It may appear very inconsequent to be told that animals very susceptible to tetanus, such as the guinea-pig and rabbit, are endowed with a natural immunity against the tetanus bacillus. And yet this fact, paradoxical as it may seem, has been demonstrated beyond doubt by Vaillard and his collaborators Vincent and Rouget[239]. When a small quantity of a culture of the tetanus bacillus was injected into one of the animals just mentioned, tetanus was not long in declaring itself. After a period of incubation, certain muscles became stiff and a tetanus, local at first, soon became general and had a fatal issue. Now, when much larger quantities of bacilli are inoculated, but care is taken to rid them of the tetanus poison elaborated in the culture-fluid, the animals resist without exhibiting any trace of tetanus. This experiment, repeated many times, always with the same result, demonstrates that the tetanus bacillus, when deprived of the co-operation of the toxin, encounters, in these animals so susceptible to the latter, a most effective opposition. Why is this? It was supposed that, in diseases like tetanus so markedly toxic in character, the resistance was in no way dependent on the phagocytic function. Thus Vaillard and Vincent were quite prepared to attribute no share to the phagocytes in the example of natural immunity which they had discovered. A detailed analysis of the facts convinced them, however, that in this they were in error. Guinea-pigs and rabbits do not contract tetanus, after the inoculation of a quantity of spores and bacilli of tetanus deprived of their toxin, solely because of the occurrence of very pronounced phagocytosis. Such an injection is soon followed by a very marked invasion of leucocytes which cram themselves with spores and bacilli without being in any way inconvenienced thereby (Fig. 37). Once the phagocytes have devoured all these organisms, the latter become incapable of producing their morbific effect. The spores cannot germinate within the phagocytes, but there undergo a marked degeneration and finally, after a longer or shorter interval, disappear.

When, on the other hand, the tetanus bacilli or their spores are accompanied by the pre-formed toxin, the latter, according to Vaillard, excites a negative chemiotaxis of the leucocytes which keep away from the organisms and which are thus allowed to multiply and to secrete fresh quantities of toxin. The natural immunity of the animal’s organism against the tetanus bacillus can be suppressed whenever the phagocytic defence is hampered in any way. Under natural conditions it is usually the adjuvant micro-organisms that aid the tetanus infection by hindering the phagocytes from seizing the spores with sufficient rapidity to prevent their germination. This fundamental result, established by Vaillard and Vincent, has often been gainsaid on the evidence of insufficient experiments (Sanchez-Toledo, Klipstein, Roncali), but, ultimately, its accuracy has been completely confirmed. Cases have been cited in which the tetanus spores, deprived of their toxin, still set up a fatal tetanus. When a small fragment of an agar culture of tetanus, previously heated to 85° C. for the purpose of destroying the toxin, is inoculated, we produce tetanus. Vaillard and Rouget have demonstrated that, under these conditions, the leucocytes penetrate merely into the superficial layer of the agar, the spores germinating and the bacilli multiplying in the deeper part. We can also set up a fatal tetanus in animals by inoculating, along with sterilised earth, spores deprived of their toxin by means of heat. The particles of soil protect the spores against the aggression of the phagocytes, allow them to germinate and then to poison the organism. Lactic acid produces an analogous effect, by destroying or weakening the phagocytes. Micro-organisms, most often inoffensive in themselves, also prevent the phagocytosis of the tetanus spores and thus aid the intoxication.

[Sidenote: [181]]

The facts above summarised have been demonstrated to be the rule for several species of anaerobic pathogenic bacteria. Thus, Besson[240] showed that the septic vibrio is, by itself, incapable of setting up septicaemia; in order to do this it needs the co-operation of other micro-organisms. Leclainche and Vallée[241] have extended the same rule to the bacillus of symptomatic anthrax (_Bacillus chauvaei_), so important as being the cause of an epizootic disease of the Bovidae. The spores of this bacillus when heated to 80°–85° C. lose the preformed toxin and at once become incapable of producing infection.

In this case also, these spores soon after injection become the prey of phagocytes, which seize them, prevent their germination and check their pathogenic action. If to these heated spores, however, we add a small quantity of toxin, they are enabled to germinate in the tissues and set up a typical infection. If heated spores are mixed with sterile sand, and the mixture introduced into guinea-pigs, these animals almost invariably acquire a fatal symptomatic anthrax. The spores in the superficial part of the sandy mass are readily devoured by the phagocytes; but those which are included within the central part of the mass, being protected for some time against these cells, germinate as soon as they become permeated with the fluids of the animal organism. If we envelope the sand in a paper sac the protection against the phagocytes is still more complete and allows almost all the spores to germinate and in every case to set up a fatal infection. Leclainche and Vallée conclude from their experiments “that we only require to protect the spore _mechanically_ in order to see an infection produced; here we cannot allege an increase of its virulence, as when we associate a chemical substance with the virus, and the exclusive part played by the phagocytosis in the protective process stands out clearly” (p. 221).

The history of these three anaerobic organisms clearly proves that the natural immunity against them cannot be made dependent on either the bactericidal power of the fluids, or on any antitoxic property, or on the incapacity of the micro-organism to secrete its toxin in the fluids of the refractory animal. The cause of this immunity resolves itself into the reaction of the phagocytes which prevent the micro-organisms from producing their poisons.

[Sidenote: [182]]

All that has been said on the subject of the natural immunity of the Vertebrates has had reference to cases of resistance against Bacteria. But may not the immunity against micro-organisms belonging to other groups depend on other factors with which the reader has not yet been made sufficiently acquainted? Amongst the lower plants there are Blastomycetes (_Torulae_ and Yeasts) which are capable of producing infections, e.g. the disease amongst the _Daphniae_.

[Sidenote: [183]]

Some observers, no doubt, have come to the conclusion that the various Blastomycetes, when introduced into a refractory organism, undergo complete destruction within a few hours without any intervention of phagocytosis. Thus Jona[242] explains the disappearance of yeast-cells injected into the veins or peritoneal cavity of the rabbit as due to the sole influence of the microbicidal property of the bloodfluid. Gilkinet[243] looks at it from the same point of view. He injected beer yeast (_Saccharomyces cerevisiae_) into a rabbit and observed that it had disappeared shortly afterwards. The destruction of the yeast-cells, according to this observer, “is effected by means of plasmatic juices” and “is due to a specific property of the organic fluids” whose nature is “quite unknown as regards its essential principle.” Phagocytosis is said to play no part in this phenomenon. Let us hasten to say that before the publication of the two works just cited, a memoir by Schattenfroh[244] had appeared on the same subject. This observer, who carried out his experiments in Buchner’s laboratory at Munich, accurately observed and described the destruction of injected yeasts by phagocytes, whilst his experiments on the microbicidal power of the blood and serum failed. This testimony is the more important that it emanates from a school by whom the microbicidal power of the “humours” is regarded as the principal factor in the defence of the animal organism. The facts described by Schattenfroh are perfectly accurate and have been confirmed in my laboratory by Skchiwan[245], who did not restrict himself to injecting ordinary yeasts (pink yeast, _Saccharomyces pastorianus_) but inoculated guinea-pigs with pathogenic yeast-cells, isolated by Curtis[246] from a case of myxomatous tumour in man. The guinea-pig is refractory to small doses of this yeast but succumbs to injections of larger quantities: Skchiwan convinced himself that the ingestion of the non-pathogenic yeast-cells takes place with great rapidity. Thus the _Saccharomyces pastorianus_, in the peritoneal cavity of the guinea-pig, is ingested almost exclusively by microphages at the end of two hours. Some (3–4) hours after injection, “sowings” of the peritoneal exudation no longer yield growths. On the other hand Curtis’ pathogenic yeast-cells resist the action of the phagocytes for a much longer time. After a period of phagolysis in the peritoneal cavity, the leucocytes that have just arrived in large numbers begin to seize the yeast-cells. Usually several macrophages fuse around the same yeast globule forming a very characteristic kind of rosette. Sometimes the macrophages run together to produce a giant cell, whose centre contains the yeast-cell. This latter defends itself against phagocytosis by secreting a fairly thick membrane. The struggle between the two living elements is a fairly prolonged one; 24 to 48 hours after inoculation all the yeasts are surrounded by phagocytes, amongst which microphages are exceptional. But the parasites remain alive for 4–6 days after their injection into the peritoneal cavity, as proved by the cultures that are obtained from the exudation when the fluid is “seeded” out. It must be concluded, therefore, that the yeast-cells were surrounded by the phagocytes whilst still presenting all the signs of life. Skchiwan was no more successful than Schattenfroh in demonstrating any kind of microbicidal action of the fluids on the Blastomycetes.

There is, consequently, no doubt whatever that the resistance of the animal organism against yeasts follows the same rules that hold in the defence against bacteria.

[Sidenote: [184]]

The animal micro-organisms are much rarer in infective diseases than are the microphytes; moreover the impossibility of obtaining cultures of them renders their investigation much more difficult. Yet there exist facts that are capable of affording us information as to the means made use of by the refractory organism against certain parasitic Protozoa. Amongst these latter the _Trypanosomae_ play a most important part. One species of this genus (_T. lewisi_) produces an infective disease in rats, especially in the grey rat (_Mus decumanus_), the blood of these rodents often containing a very large number of them, whilst the small flagellated organisms flourish well in the serum prepared from the blood of affected animals. Laveran and Mesnil[247], in their studies on the _Trypanosomae_, injected defibrinated blood containing numerous _Trypanosomae_ into the peritoneal cavity of guinea-pigs, which exhibit a natural immunity against this parasite. The parasites remained alive for some days and then disappeared completely. Here again it is the phagocytes of the peritoneal exudation which rid the animal of the _Trypanosomae_ by ingesting them. Laveran and Mesnil were able, by the examination of hanging drops of the peritoneal exudation of their guinea-pigs, to detect leucocytes in the act of devouring _Trypanosomae_ which showed, by their active movements, that they were still alive. Once the parasites were completely enclosed within the macrophages, their final disappearance took place with extraordinary rapidity.

In this chapter we have attempted to place before the reader a complete series of the phenomena observed in natural immunity in animals. We have passed in review the resistance of the animal organism against the principal groups of bacteria, and we have dwelt on certain of them which are most capable of adapting themselves to various media, and on others which present examples of microorganisms more exacting and more delicate. We have examined the immunity against Blastomycetes and parasitic animalcules. Above all, in the lower animals, just as in the Vertebrata of all classes, we have always observed this general phenomenon: phagocytic resistance as the principal and constant factor in natural immunity.