CHAPTER VI

NATURAL IMMUNITY AGAINST PATHOGENIC MICRO-ORGANISMS

Natural immunity and the composition of the body fluids.—Cultivation of the bacteria of influenza and pleuro-pneumonia in the fluids of refractory animals.—Resistance of _Daphniae_ to the Blastomycetes.—Examples of natural immunity in Insects and Mollusca.—Immunity of Fishes against the anthrax bacillus.—Immunity of frogs against anthrax, Ernst’s bacillus, the bacillus of mouse septicaemia and the cholera vibrio.—Natural immunity in the cayman.—Immunity of the fowl and pigeon against anthrax and human tuberculosis.—Immunity of the dog and rat against the anthrax bacillus.—Immunity of Mammals against anthrax vaccines.—Immunity of the guinea-pig against spirilla, vibrios, and streptococci.—Natural immunity against anaerobic bacilli.—Fate of Blastomycetes and _Trypanosomae_ in the refractory organism.

[Sidenote: [136]]

In the third chapter reference has been made to the frequency of cases of natural immunity against infective diseases. Examples of this immunity occur in the lower animals—the Invertebrata—and are widely met with among the Vertebrata. We have already mentioned that this natural immunity can be attributed neither to insusceptibility to microbial toxins nor to the elimination of the micro-organisms by the excretory channels. Nevertheless the pathogenic agents which have penetrated into the tissues of the refractory organism disappear, without being eliminated. To facilitate the study of their disappearance it has been necessary to pass in review the phenomena that follow the introduction of foreign bodies into the organism and to present a brief analysis of the process of resorption of cell elements in its relations to digestion. We have tried to demonstrate that resorption is nothing more than a process of digestion which, instead of going on in the intestinal canal, takes place in the tissues; that it is, indeed, an intracellular digestion exactly comparable to that which serves for the nutrition of certain of the lower animals.

[Sidenote: [137]]

A knowledge of all these facts is necessary before we can deal with the subject to which the present chapter must be devoted—the innate natural immunity of animals and man against pathogenic micro-organisms. As, under natural conditions, it is the micro-organism and not its toxic products which invades the organism, it is clear that we must give the first place to the study of immunity against the micro-organism. The more so because this form of immunity is much more frequently met with than is an insusceptibility to toxins.

Since the animal organism has a very variable composition it might be concluded that the micro-organisms find in the refractory species simply a chemical medium in which they cannot live. We cannot go far in the discussion of this supposition without seeing that it may be rejected. Among the pathogenic micro-organisms some are distinguished by a great fastidiousness and sensitiveness as regards the medium in which they are placed. Such, for example, are the parasites of malaria and their allies. They live inside the red blood corpuscles of Vertebrata and appear to be extremely discriminating in regard to their requirements. All animals, even monkeys, are refractory to human malarial fevers. It might be concluded from this that here at least the immunity may be due to the fact that the chemical composition of the contents of the red corpuscles in the immune animals is different from that of the red corpuscles of man. But when we see, as was first demonstrated by Ross[175], that the malaria parasite of Laveran, having made its way into the digestive canal of certain mosquitos (_Anopheles_), there develops abundantly, it is difficult to maintain this thesis.

Among other micro-organisms of animal origin we have the _Trypanosoma_, the parasite of the terrible disease propagated by the Tsetse fly which commits such ravages amongst mammals. Man alone escapes it, exhibiting a natural immunity that nothing apparently can overcome. Are we to affirm that it is the difference in the chemical composition of the human body which assures to man his immunity against a parasite that attacks indifferently an herbivorous animal, such as the ox or rabbit, or a carnivorous animal, such as the dog? In these examples I have chosen merely those micro-organisms which it has never been possible to cultivate on any artificial nutrient medium and which are kept alive with very great difficulty outside the living organism.

[Sidenote: [138]]

What is to be said then of the vegetable micro-organisms which, in this respect, are much less exacting? The most important of these and the most numerous of all pathogenic micro-organisms, the Bacteria, can as a rule be cultivated without difficulty not only in the blood and fluids of animals that are susceptible or refractory to their morbific action, but also on all kinds of vegetables and artificial media: broths, fluids composed of mineral salts and of certain organic substances. It is really not possible to attribute the natural immunity of the dog and the fowl against the anthrax bacillus—so fatal to a great number of mammals, man included,—to its incapacity to feed on the fluids of these animals, when we see that this same bacillus is capable of killing lower animals, such as the cricket, and can thrive on carrots, potatoes and other vegetables.

Even when, among the bacteria, we take those that are most exacting in the choice of their food, we still find it impossible to explain natural immunity as being due to the want of power on the part of these organisms to obtain their nutriment from the juices of refractory species. The bacillus discovered by R. Pfeiffer[176] in influenza does not develop on media that are ordinarily employed in bacteriology in the cultivation of a great number of micro-organisms. It needs a special food, which is prepared for it by spreading a drop of fresh blood on the surface of agar. Pfeiffer has established the fact—confirmed by many observers—that the best species of blood to use for this purpose is that of the pigeon. We should have to believe, then, did the immunity really depend on the composition of the fluids, that the pigeon is the least refractory of all animals. Experiment has demonstrated the erroneousness of such a supposition: the pigeon is quite as refractory to Pfeiffer’s bacillus as are most other species of animals.

[Sidenote: [139]]

As a second example the bacterium of bovine pleuro-pneumonia may be cited. It is the smallest of all known bacteria. The difficulties surrounding the discovery and identification of this organism were very great, and the ingenuity of Nocard and Roux[177] was required for the demonstration of its existence. Very exacting in its choice of nutritive material, it was first cultivated in the fluids of the rabbit, a species endowed with an absolute immunity against bovine pleuropneumonia. It is unnecessary to multiply examples to obtain a general proof that natural immunity against micro-organisms cannot be explained by the incapacity of these pathogenic agents to live in the fluids of the refractory organism.

We must, however, ascertain what takes place in resistant animals inoculated with micro-organisms. Here, again, it is preferable to begin with the lower animals of simple organisation. We have already seen that examples of natural immunity are not rare in the Invertebrata. When engaged in the study of the disease found in _Daphniae_, small crustacea so common in fresh water, I was able to show that the special Blastomycetes which cause it meet with a vigorous resistance on the part of the organism. As the _Daphniae_ are small, transparent, and consequently easily observed under the microscope, I was able without difficulty to establish the main phenomena observable in these organisms. I can be the more brief in describing these phenomena of resistance as, in addition to devoting a special memoir to the _Daphnia_ disease[178], I have, in my _Lectures on Inflammation_ (pp. 97–103)[179], described at some length the reaction of their organism to the _Monospora_. It is nevertheless necessary that I should recall, very briefly, the mechanism by which these small crustaceans secure immunity.

The spores of the parasite—very delicate and rigid needles—are swallowed with the food. By means of their sharp points they perforate the intestine and penetrate into the body cavity, full of blood, where they find themselves exposed to the attacks of leucocytes. These leucocytes, guided by their tactile sense, gather around the foreign body, ingest it completely and destroy it. It is remarkable that the spore, which is furnished with a very resistant membrane, once in the interior of the mass of leucocytes, undergoes modifications which afford evidence of the presence in these cells of an extraordinary digestive power. The surface of the spore, from being smooth and regular, becomes pitted and sinuous, the spore breaks up into fragments and is reduced to a mass of _débris_ which, in the form of brown granules, remains indefinitely in the contents of the leucocytes. From this it is evident that these phagocytes must produce a ferment which is capable of digesting the cellulose or analogous substance which forms the membrane of the spore. Unfortunately, the small size of the _Daphniae_, so useful for the direct observation of the phenomena of immunity, presents an insurmountable obstacle to the study of its leucocyte ferments, especially _in vitro_.

[Sidenote: [140]]

The destruction of the spores of the parasite by the leucocytes secures to the _Daphnia_ a real immunity. Of a hundred _Daphniae_ taken in my aquarium and carefully examined under the microscope, fourteen only were found to be infected by the budding conidia of the parasite, whilst fifty-nine of the others contained the remains of spores that had been destroyed by the phagocytes. When transferred to pure water containing no new source of contagion, these _Daphniae_ flourished and lived a normal life, giving birth to a numerous progeny.

The immunity of the _Daphnia_, due to the intervention of phagocytes, is an example of natural, individual immunity. It is not the specific or racial possession of these crustacea, for when the leucocytes do not seize the spore, at once, on its penetration into the body cavity, it commences to germinate and gives rise to a whole generation of budding cells. These cells, then, secrete a poison which not only repels the leucocytes, but kills and completely dissolves them. Under these conditions the _Daphnia_ is disarmed; the parasites grow in the organism, deprived of its arm of defence, as in a culture tube, and the animal rapidly succumbs.

Since I first observed this struggle between the _Daphnia_ and its parasite, some eighteen years ago, no other example has been found that is so easily observed and so demonstrative of the protective action of phagocytes in an animal that can be kept under observation, alive, under the microscope. Cases, however, are not wanting in the Invertebrata in which the different phases of this struggle may be observed with sufficient accuracy to warrant the conclusion that in these cases also the phenomena are analogous to those observed in the case of the _Daphniae_.

[Sidenote: [141]]

It has already been stated in Chapter III. that the larvae of the rhinoceros beetle (_Oryctes nasicornis_), although very sensitive to the cholera vibrio, are very refractory to anthrax and diphtheria. In order that we may obtain some idea of the mechanism of this immunity let us inject into the body cavity of these large white grubs a trace of anthrax culture. In the blood, drawn off the following morning, the injected bacilli are found, not in the plasma, but inside many of the leucocytes. Here there has occurred, as in the _Daphnia_, an ingestion of the parasites which have then been destroyed by the intracellular digestion of phagocytes. The process is the same, then, as that by which the resorption of the red corpuscles of the goose takes place when they are injected into the blood of cockchafer larvae. In both cases the foreign bodies are ingested and destroyed by the leucocytes of the blood; this act of resorption, however, taking a very long time.

Although the leucocytes of the larvae of the rhinoceros beetle exhibit a positive chemiotaxis for the bacillus, these same cells behave in a very different fashion in presence of the cholera vibrio. Very small quantities of this vibrio, when injected into the blood of the larvae, give them a fatal disease: the vibrios excite in the leucocytes a negative chemiotaxis and flourish without hindrance in the blood plasma. The larva is soon transformed into a culture vessel and the numerous vibrios that develop in it cause the death of the animal.

The difference in action of the two bacteria cannot be explained by any corresponding difference in their mode of life in the blood. Removed from the organism the blood plasma of the white larvae of the rhinoceros beetle is a culture medium just as favourable to the growth of the anthrax bacillus as to that of the cholera vibrio. Moreover, the former of these micro-organisms is quite capable of setting up a fatal disease in other representatives of the class of Insects. Kovalevsky[180] has discovered in the house cricket four phagocytic organs, with a great appetite for all kinds of foreign particles that may penetrate into its body. The blood of mammals, when injected below the skin of the cricket, is rapidly absorbed by the cells of the four “spleens” (for so Kovalevsky designates these phagocytic organs). The resorption of the red blood corpuscles goes on within these phagocytes owing to their power of intracellular digestion. When Kovalevsky kept crickets at a temperature of 22°–23° C. and injected them with anthrax bacilli he noted that these bacilli also were ingested by the cells of the spleens. There was, thus, no manifestation of negative chemiotaxis of these elements towards the bacillus. The ingestion of the bacilli by the phagocytes was not sufficient, however, to protect the animal. The bacilli reproduced themselves rapidly in the blood fluid; the intracellular lacunae of the spleens were full of them and the crickets quickly succumbed to the infection.

[Sidenote: [142]]

Nevertheless these crickets are quite capable of resisting certain other bacteria. Balbiani[181] has shown that they are refractory to a great number of bacilli belonging to the group of _Bacillus subtilis_. He observed that when injected into the body of the cricket these bacilli are devoured and destroyed by the leucocytes of the blood and by the large cells of the pericardial tissue corresponding to the elements of the spleens of Kovalevsky. Whilst the crickets and other Orthoptera, which are rich in phagocytes, exhibit a real immunity against these bacilli, insects which have very few leucocytes such as butterflies, flies and Hymenoptera are found to be much more susceptible to infection by the same bacilli. In this case the direct relation between immunity and phagocytosis is very marked.

[Sidenote: [143]]

The Mollusca also furnish some interesting examples of natural immunity. Karlinsky[182] has observed that anthrax bacilli, when injected into the blood of slugs and snails, soon disappear from their bodies; these pulmonate Gasteropods are absolutely unaffected by this bacillus so formidable for many species of animals. From the rapidity of this disappearance of the bacilli it has even been concluded that it was impossible for this bacillus to live in the fluids of Mollusca. Kovalevsky (_l.c._ p. 443) has studied this question with the carefulness that characterises all his work. He confirms the fact that snails (_Helix pomatia_) resist the introduction of a large quantity of anthrax bacilli into their bodies; he notes also that these bacteria disappear from the blood. But he finds them again in the tissues of the foot, and especially in the cells which surround the pulmonary vessels. “The greater number of the bacteria are found in the cells of that part of the pulmonary region in _Helix_ which adjoins the heart and kidney. All the bacteria were ingested by the cells and I easily succeeded in demonstrating this not only in sections but also in bulk” (p. 444). The snails remained in good health in spite of the presence in their phagocytes of numerous bacteria which maintained themselves there for some time. At the end of ten or twelve days and more these bacteria still presented their usual aspect; this accords well with the slowness with which intracellular digestion goes on in the majority of the Invertebrata. These bacteria were, however, no longer living, although still undigested. Morsels of the pulmonary tissue of the snails that were injected with anthrax bacilli still gave cultures 48 hours after injection and contained bacilli capable of giving fatal anthrax to mice. Later, media seeded with similar particles remained sterile, and mice inoculated therewith continued to live. From these experiments it may be accepted that bacteria, living in the blood plasma, become the prey of phagocytes which render them inoffensive and kill them. This example demonstrates once again that the organism gets rid of bacteria by the same mechanism as that which serves for the resorption of any of the formed elements. The snail reacts to bacteria as it does to the red corpuscles of the goose.

It is unnecessary to insist further on the natural immunity of the Invertebrata, and it is useless to multiply examples which always point in the same direction: to the importance of phagocytic reaction and of intracellular digestion in resorption and immunity. We must pass on to the examination of the reaction phenomena of the vertebrate organism towards pathogenic micro-organisms, following, as hitherto, the comparative method. We will commence with the study of the natural immunity of fishes as lower representatives of the great group of the Vertebrata.

[Sidenote: [144]]

It is well known that fishes are liable to infective diseases and pisciculture has often to deplore considerable losses brought about sometimes by certain of the lower Fungi (_e.g. Saprolegniae_), sometimes by Bacteria. The pathogenic microbes which produce epidemics in fishes are still little understood; but among the bacteria which kill many of the higher animals are some which cause fatal maladies in certain fishes. Thus the anthrax bacillus so virulent for many mammals is capable also, as we have seen, of producing an infection in the cricket, and may cause the death of small marine osseous fishes, the _Hippocampi_. Sabrazès and Colombot[183], who have studied this question, have demonstrated that the anthrax bacillus, which is virulent for the rabbit, when inoculated into these fishes first produces swellings at the seat of inoculation and ultimately becomes generalised throughout the body, producing a fatal septicaemia. As these experiments have given this result at a temperature of 14°–16° C., it is quite evident that the bacillus, in order to manifest its pathogenic effect, in no way needs the high temperature of the mammalian body for its action.

Now among fishes there are not wanting species which resist the anthrax bacillus. Mesnil[184] has, in our laboratory, thoroughly studied the mechanism of this immunity. He has shown that several fresh-water fishes, _e.g._ the perch (_Perca fluviatilis_), the gudgeon (_Gobio fluviatilis_), and the gold-fish (_Carassius auratus_), will resist an injection of a considerable number of bacilli into the abdomen. When kept at temperatures of 15°–20° C. or even 23° C., a temperature at which the bacilli are able to develop very abundantly, these fishes destroy a large number of the bacteria in their bodies. Soon after the introduction of the bacilli into the peritoneal cavity, the numerous leucocytes accumulate around them and ingest them by the same mechanism that is observed in the Invertebrata or in the same fishes when absorbing the red blood corpuscles of alien species. In the gudgeon, at as early as six and a half hours, a very marked, nay, an almost complete phagocytosis is set up.

It is impossible to doubt the fundamental fact that the bacilli, at the moment of their ingestion, are in a perfect condition of vitality and virulence. The fluid of the peritoneal exudation, when withdrawn from the animal, is of itself incapable of preventing the development of the anthrax bacilli. The peritoneal lymph of the above-mentioned fishes is, _in vitro_, even a good culture medium for these bacilli.

[Sidenote: [145]]

When, long after the completion of the phagocytosis by the leucocytes of the peritoneal exudation, a drop of the exudation is withdrawn and kept outside the organism under suitable conditions of temperature and moisture, a number of the ingested bacilli begin to multiply and give an abundant culture. This experiment proves, indisputably, that the bacilli are devoured in the living state. If a little of the peritoneal exudation, withdrawn several (up to nine) days after the injection of the bacilli, be injected below the skin of guinea-pigs these animals die from generalised anthrax, a fact which demonstrates that the bacilli, which have been ingested alive, have retained their virulence a long time after they have been devoured by the leucocytes. But, if the peritoneal exudations that have been withdrawn at still longer periods after injection be examined, it is found that they no longer contain bacilli capable of developing in culture media or of setting up the disease in the most susceptible animal. Hence it follows that in the organism of the refractory fish, the bacteria are not destroyed by the fluids but by the phagocytes, which take a long time to bring about the complete intracellular digestion of ingested micro-organisms.

The phagocytes which assure immunity to the osseous fishes that were studied by Mesnil belong principally to the group of haemomacrophages. These are leucocytes with abundant protoplasm which stain readily by basic aniline dyes, mononuclear cells whose nucleus, however, is sometimes divided into lobes. It is to be noted that in the perch these are the sole representatives of the motile phagocytes, and that in this fish not only the eosinophile but every other variety of granular leucocyte is completely wanting. In the gudgeon, in addition to haemomacrophages, some microphages whose protoplasm stains faintly with acid aniline colours are met with. These facts will be useful to us when we come to study the part played by phagocytes in immunity from a general point of view.

Another class of cold-blooded animal, the Amphibia, has been much more frequently studied from the point of view of infection and immunity. The frog, an animal so convenient for many physiological and pathological researches, has been much employed for the study of immunity against pathogenic micro-organisms. Quite a literature, which has been excellently summarised in the memoir of Mesnil already cited, and to which we shall have occasion to return more than once, has been accumulated on the subject.

[Sidenote: [146]]

The immunity of frogs against the anthrax bacillus was early demonstrated and studied in Robert Koch’s celebrated memoir[185] on anthrax. This observer, after injecting an emulsion of anthrax spleen into the lymph sac of the frog, recovered the bacilli from the interior of round cells which burst readily when transported into water. Koch, accepting the view then generally held, thought that the bacilli found a favourable culture medium in the contents of certain cells, but that, in spite of this, the frog was capable of manifesting a real immunity against anthrax. Gibier[186] made the interesting discovery that frogs when subjected to the influence of high temperature (about 37° C.) lose their natural immunity and readily contract fatal anthrax.

Since that time the mechanism by which the organism of the frog secures immunity against the anthrax bacillus has repeatedly been studied. In a memoir which appeared in 1884[187] I insisted that the principal part played in this immunity belonged to the phagocytes which devour the injected bacteria and subject them to intracellular digestion. The round cells described by Koch are nothing but the leucocytes of the lymph sac which have seized upon the anthrax bacilli. These bacilli instead of thriving in the cell contents find there a very unfavourable medium, and perish at the end of a longer or shorter period. When the activity of the phagocytes is impeded by unfavourable influences, _e.g._ high temperature, they exhibit a very feeble reaction, incapable of assuring to the frog that immunity which, under normal conditions, it possesses. The conclusions I have just summarised have raised very lively opposition from a large number of observers. Baumgarten[188], with his pupils Petruschky[189] and Fahrenholtz[190], have endeavoured to demonstrate that phagocytosis plays no part in immunity and that the frogs resist anthrax simply because the bacilli are incapable of maintaining themselves alive in the fluids of this Batrachian. Nuttall[191], of Flügge’s school, also maintained that frogs resist anthrax owing to the bactericidal power of their fluids. This view has been defended by several other observers and appeared for some time to become quite dominant.

[Sidenote: [147]]

Nevertheless, it is possible to demonstrate that the plasmas of the frog not only are not inimical to the life of the bacillus, but serve as a good culture medium for it[192]. All that is necessary for the demonstration of this fact is to introduce below the skin of frogs anthrax spores enclosed in a sac of reed pith, or simply enveloped in a small piece of filter paper. The plasma of the lymph sac at once permeates the spores and allows them to germinate and produce quite a generation of bacilli. But, as soon as the leucocytes pass through the paper, they seize upon the young bacilli, digest them in their substance and prevent their pathogenic action. The germination of the spores may take place even where they have been introduced below the frog’s skin without being protected in any way whatever. But, under these conditions, only a certain number of the spores germinate, the majority not having time to do so before the arrival of the leucocytes. The small, very short bacilli which proceed from the germinated spores, are, along with the spores that have not germinated, soon ingested by the phagocytes. But, whilst the rods are in the end digested within these cells, the ingested spores remain intact for a very long time: they do not germinate, but they are not destroyed and retain their vitality indefinitely, in spite of the influence of the phagocytes. It is sufficient to withdraw from a frog, that has been inoculated with anthrax spores some time before and kept at a moderate temperature (15°–25° C.), a little lymph and sow it in any nutrient medium (of those employed in the culture of bacteria), in order to see the spores germinate and produce a whole generation of absolutely normal filamentous bacilli. All these phenomena have been carefully studied by Trapeznikoff[193] in a work executed in my laboratory. It is obvious from his experiments that the phagocytes of the frog are quite capable of protecting the organism against the anthrax bacillus by ingesting and digesting the bacilli in the vegetative state and by preventing the germination of the ingested spores. This phagocytic action is very important in presence of the fact that the plasmas of the frog allow the spores to germinate and the bacilli to develop and produce abundant cultures.

[Sidenote: [148]]

The immunity of frogs against the anthrax bacillus that we have just described and which is guaranteed by the activity of the phagocytes, is constant under the conditions of temperature above mentioned (15°–25° C.), conditions which are sufficient, however, to ensure the death of susceptible cold-blooded animals, such as the cricket or _Hippocampus_, from anthrax. The edible frog, a species that readily accommodates itself to a temperature of 35° C., resists, even under these conditions, infection by the bacillus, as pointed out by Mesnil in a work already cited when treating of the immunity of fishes. The green frog (_Rana esculenta_) when kept for a long time at this high temperature, so suitable for the development of the anthrax bacillus, reacts by the same phagocytic mechanism. The leucocytes of the lymph and blood, the cells of the splenic pulp and Kupffer’s stellate cells of the liver, seize the introduced bacilli and digest them as in any other case of phagocytosis. The brown frog (_Rana temporaria_) adapts itself but slightly and with great difficulty to the high temperature and dies whether it has been inoculated with anthrax or not. Under these conditions the bacteria develop in the body of the dead or dying frogs, but Mesnil insists on the fact that a true anthrax infection is not produced, as has been maintained by Gibier as the outcome of his researches.

[Sidenote: [149]]

Dieudonné[194], however, has found a method of removing the natural immunity of the frog against the anthrax bacillus, by inoculating it with an artificial bacterial race which he had adapted to develop fairly luxuriantly at the low temperature of 12° C. Under these conditions all the inoculated frogs, even those which had resisted the inoculation with ordinary bacteria (grown at 37°·5 C.), died within a period of 48 to 56 hours, containing many bacilli in the blood and organs. Dieudonné has not studied the essential mechanism that accompanies this loss of immunity; but it is very probable that, for one thing, we have here to do with a reinforcement, special for the frog, of the bacillus that has become accustomed to develop at a low temperature. This bacillus must multiply, in frogs that have been maintained at a low temperature, much more rapidly and profusely than would the ordinary bacillus. On the other hand, the susceptibility of Dieudonné’s frogs must depend on a less resistance of the organism under the conditions of his experiments. Unfortunately, we cannot find in his memoir sufficient data on these points; he does not even state the temperature at which the frogs that had been inoculated with bacteria adapted to cold lived. Dieudonné invokes the analogy of his results with those obtained in the case of the immunity and susceptibility of frogs as regards a septicaemic bacillus.

This bacillus (_Bacillus ranicida_) has been made the subject of an interesting study by Ernst[195]. It is a small, very slender bacillus, which, in frogs, produces a fatal malady epidemic in spring, but ceasing completely during summer. Taking this fact as a basis, Ernst has succeeded in conferring immunity upon frogs in autumn by placing them in an incubator at 25° C. In spite of the injection of a considerable dose of the small bacillus, the frogs living at this temperature remained in good health, whilst control animals exposed to a low temperature died of septicaemia. The counter-test was made in summer. Inoculated frogs that were kept in the laboratory were unaffected, whilst those that had been kept in a refrigerating apparatus at 6°–10° C. invariably died. It may be asked, Is this evident influence of temperature on immunity and receptivity exercised on the organism of the frog or upon the pathogenic bacillus? In the case where a bacillus can only develop at low temperatures its harmlessness at the higher temperature may be readily understood. The experiments of Ernst have demonstrated, however, that this small bacillus develops much better at 22° C., and even at 30° C., than at lower temperatures. It must be concluded, therefore, that the high temperature which confers immunity acts not by weakening the bacillus, but rather by reinforcing the resisting power of the organism. The low temperatures (6°–10° C.) that are favourable to a fatal infection have a different action; that is to say, they weaken the reaction of the inoculated frogs.

[Sidenote: [150]]

Although Ernst has not studied the mechanism of this resistance fully, it is evident, from the data he has supplied, that it consists in a phagocytic reaction. He was able to demonstrate the ingestion of the bacilli by the phagocytes in the susceptible refrigerated frogs, as well as in the refractory frogs, kept at a higher temperature; but in the former case the phagocytosis was so feeble that 24 hours after inoculation a considerable number of free bacilli were still found in the lymph of the dorsal sac, whilst in the refractory frogs the much more active phagocytosis brought about the disappearance of the free bacilli during the first day. If, as is very probable, the analogy of this septicaemia with anthrax in frogs, upon which Ernst insists, really exists, it must be concluded that the susceptibility of these Batrachians to the modified race of the bacillus depends on their weak phagocytic resistance.

Since, in these two examples of natural immunity in the frog, we have seen that the phagocytic activity exhibits itself in an active form against bacteria which readily develop in the fluids of the same animal, we might conclude that the reaction of the phagocytes constitutes a general mode of defence in cold-blooded animals. But Lubarsch[196], a very cautious observer, has expressed an opposite view, based on his studies on the bacillus of mouse septicaemia. He convinced himself that frogs will resist injections of even considerable quantities of this bacillus, without any co-operation on the part of the phagocytes. As we have, here, to do with a matter of fact, Mesnil (_l.c._) set himself to verify these observations, with the object of establishing whether it was a case of a real exception or of a simple misunderstanding. He was able to demonstrate, by irrefutable observations and experiments, that the bacilli of mouse septicaemia when inoculated into frogs, set up a very pronounced positive chemiotaxis on the part of the phagocytes, which seized and digested the bacilli just as they do the anthrax bacillus. This apparent exception, therefore, becomes transformed into an additional argument in favour of phagocytic reaction being a general factor in immunity. In support of this hypothesis I may adduce a further example, already mentioned in a preceding chapter when discussing another question. The frog is very refractory against the cholera vibrio. When these vibrios are inoculated into the dorsal lymphatic sac or into any other part of the body the animal retains its health unimpaired. An examination of the exudation at the point of inoculation demonstrates that the vibrios meet with a vigorous opposition on the part of the phagocytes, which ingest and completely digest them. This is of special interest from the fact that the frog is very sensitive to the toxin of the cholera vibrio. When injected in a weak dose it kills the frog very quickly. Two small frogs died in less than an hour from the effect of 0·5 c.c. of cholera toxin.

[Sidenote: [151]]

The natural immunity of the frog against the cholera vibrio affords, then, an example in which the organism, destroying the vibrio by phagocytosis, prevents the production of the poison, which, otherwise, would infallibly kill it.

Having demonstrated that phagocytic reaction manifests itself in the frog in all cases of natural immunity that have been sufficiently studied, we must dwell for an instant on the question of the condition of the bacteria at the moment of their ingestion by the phagocytes. It is very evident that this phagocytic defence is only efficient on condition that it is exercised against bacteria which, in its absence, might injure the organism by their multiplication and their virulence. For this reason the question as to whether the micro-organisms, before being ingested, were living and capable of producing their pathogenic action has been widely discussed. It has even been suggested that the phagocytes are only capable of ingesting the dead bodies of micro-organisms that have been killed by other agents. Frogs are very suitable for a study of this question. When a drop of the exudation is removed some time after inoculation with a motile organism, such as the _Bacillus pyocyaneus_ or the cholera vibrio, the organism was often found moving rapidly within the vacuoles inside leucocytes. The experiment will succeed even more completely if a drop of frog’s lymph be mixed, on a slide, with a trace of a culture of these motile micro-organisms, the latter being soon found in the clear vacuoles included in leucocytes and executing extremely rapid movements.

Besides this direct proof we can assure ourselves of the living condition of the micro-organisms in another way. Withdraw a drop of the exudation at an advanced stage of the process when there are no longer any free micro-organisms; inside the phagocytes a few scattered bacteria, more or less well preserved, can still be seen. It is sufficient to keep a hanging drop of such an exudation at a temperature of about 30° C., care being taken to keep it from drying, but without adding to it any nutrient medium. Under these conditions the leucocytes die more or less rapidly, but the bacteria regain vigour: they begin to multiply, and at the end of a short time produce a generation of bacteria within the dead leucocyte. The multiplication of the bacteria goes on progressively and the hanging drop is transformed into a real pure culture. Mesnil was able to confirm these data with the exudations of frogs that had been inoculated with either the bacilli of anthrax or of mouse septicaemia.

[Sidenote: [152]]

The bacteria, ingested in the living state by phagocytes, retain their original virulence. Some authors think, and I was formerly of this opinion, that at the end of a more or less prolonged sojourn within the leucocytes, anthrax bacilli undergo an attenuation in their virulence. Later, numerous researches have, however, demonstrated that this opinion is incorrect, and that the virulence is maintained in the bacteria included in the phagocytes of frogs the whole time that these bacteria remain alive. Dieudonné has insisted on this fact as regards the anthrax bacillus. Mesnil has confirmed it for this same species and for the bacillus of mouse septicaemia. It is impossible, therefore, to doubt this general result, that frogs which are refractory against certain bacteria resist because of the phagocytosis which is exercised against living and virulent micro-organisms.

We have insisted sufficiently on the analysis of the natural immunity of the frog, and need not tarry over the facts relating to other amphibia which, moreover, have been much less studied. The reptiles, those higher representatives of the Vertebrata called cold-blooded, often present examples of really remarkable immunity. Thus alligators will resist enormous doses of various bacteria, such as the anthrax bacillus, that of human tuberculosis or the cocco-bacillus of typhoid fever. When, some time after an injection is made, the exudation at the point of inoculation is withdrawn there is found a large number of leucocytes, amongst which may be recognised many eosinophile microphages, though the majority are macrophages with one, two or more nuclei. Really giant cells are found in the exudation. It is the macrophages which specially manifest phagocytosis and they are often found crammed with the injected bacteria, as I was able to assure myself after injections of typhoid cocco-bacilli. The natural immunity of alligators (_Alligator mississipiensis_) persists not only at the temperature of the incubator (37° C.), but also at room temperature (20°–22° C.).

[Sidenote: [153]]

Passing in review the animal kingdom we must pause for a moment to consider the natural immunity of birds or lower warm-blooded Vertebrates. The classic example of this immunity is that of the fowl against anthrax. It has long been known that birds resist inoculation with anthrax or only exhibit a feeble receptivity; though smaller birds are for the most part susceptible to anthrax, the pigeon is much less so and the fowl presents a case of the most pronounced immunity. It was believed to be absolutely refractory until the experiments of Pasteur and Joubert[197], who found a sure method of suppressing this immunity. Fowls that had been inoculated with the bacillus were immersed up to the thighs in cold water in order to bring down their temperature. It was found that, under these conditions, the anthrax bacillus developed at the seat of inoculation and later became generalised in the blood, and invariably caused death. It was concluded from this that the natural immunity of the fowl was dependent on its very high normal temperature (41°–42°) which interfered with the pathogenic functions of the anthrax bacillus.

Hess[198] studied the mechanism of this immunity of the fowl and pointed out the important part that phagocytosis plays in the destruction of the inoculated bacteria.

These researches were resumed in my laboratory by Wagner[199]. Having established that the anthrax bacillus develops readily in the blood and the blood serum of fowls, outside the organism, at high temperatures (42°–43° C.), he came to the conclusion that the lowering of the temperature of the body of the fowls by immersing them in water produced, not a reinforcement of the bacillus, but a weakening of the resisting power of the animal. He was able to convince himself that this resistance exhibits itself in the activity of the phagocytes which ingest and destroy the anthrax bacillus in its vegetative state. In the normal fowl the phagocytosis is rapid and very pronounced, whilst in a fowl that has been refrigerated this reaction is very slight or absent. To corroborate this general conclusion, Wagner, instead of lowering the temperature by means of cold water, made use of antipyrin and chloral. The application of this treatment likewise caused enfeeblement of the natural defence of the organism and suppressed the immunity of the fowl against anthrax.

[Sidenote: [154]]

Trapeznikoff[200] has studied carefully the fate of anthrax spores when injected into fowls. He observed that most of them are devoured by the leucocytes. Some of the spores were first transformed into small rods, sometimes growing into real bacilli, but finally they all became the prey of phagocytes and perished in their interior. Those in the vegetative condition are soon digested, the spores, however, persist for some time inside the phagocytes, but ultimately disappear. The phagocytosis in fowls inoculated with spores is very marked, and preparations, stained by Ziehl’s method, demonstrate most clearly the reality of this reaction phenomenon. These preparations have for long been used in the course in bacteriology at the Pasteur Institute for the demonstration of phagocytosis.

[Sidenote: [155]]

In the face of these facts, well established and confirmed many times, it is impossible to accept Thiltges’[201] denial of the ingestion of these bacteria by the phagocytes of the fowl. Some fault of technique, which I am not at the moment in a position to indicate exactly, has evidently slipped into this author’s work. The positive data, however, on phagocytosis in the fowl, obtained by Hess, Wagner, and Trapeznikoff, data confirmed by myself, render unnecessary any fresh researches for the purpose of explaining the negative results obtained by Thiltges. As regards his experiments on the bactericidal action of defibrinated blood and of blood serum of fowls against the bacillus and its spores, experiments whose results are opposed by those of Wagner, the contradiction may be explained pretty easily, at least in part. Thiltges mentions several times that the bacilli, when sown in the blood serum of the fowl, were aggregated in clumps. Nevertheless, he has failed to guard against this source of error and has attributed the diminution in number of the colonies on plates to the destruction and not to the agglutination of the bacilli. Thiltges gives so few particulars of the conditions under which his experiments were performed that we do not even know at what temperature he kept his tubes containing blood and serum sown with bacilli. As Wagner kept his at 42°–43° C., a temperature which corresponds to that of the body of the fowl, I asked M. Gengou to make a series of experiments on the bactericidal power of the plasma and blood serum of fowls on the anthrax bacillus, keeping his tubes at 37° C. The result of his experiments was in complete accord with those of Wagner. Under the conditions that I have just stated the fluids of the fowl are no more bactericidal than they are under the conditions maintained in Wagner’s experiments.

In summing up these data on the natural immunity of fowls against anthrax, we are certainly justified in concluding that it is due to the phagocytosis and not to any bactericidal property of the “humours.”

The pigeon is more susceptible than the fowl to the action of the anthrax bacillus, still it manifests a certain degree of resistance against the microbe. After what we have said on the subject of the fowl we need make but few remarks on the pigeon, in spite of the very animated discussions that have taken place on the mechanism of its immunity. When Baumgarten was offering a systematic opposition to the part played by phagocytic reaction in immunity, he set his pupil Czaplewski[202] to investigate the resistance of pigeons against anthrax. The results of this investigation were absolutely negative as regards phagocytosis. The latter was said to have no importance in the defence of the organism, which resisted simply because it was impossible for the bacillus to live in the body of the pigeon. I then set myself to study this question[203], and I was able to demonstrate that the anthrax bacillus is quite capable of keeping alive in the pigeon, that it can develop in its fluids, but that it is unable to defend itself against the aggression of the phagocytes which ingest it and completely digest it. By isolating the phagocytes that had ingested the bacilli injected into the body of the pigeon, I was able to prove that a number of these bacilli were still alive. The enfeeblement and death of the phagocytes when outside the body allowed the anthrax bacilli again to get the upper hand in this struggle, to develop and to give virulent cultures. The part played by phagocytes in this example of natural immunity was thus placed beyond doubt.

[Sidenote: [156]]

Later, Czaplewski[204] himself became convinced that his previous negative results would not stand criticism, and Thiltges, in his work already mentioned, when discussing the fowl, was able to confirm the importance of phagocytosis in the defence of the organism of the pigeon against anthrax. He was struck by the difference between these two species of birds. In the pigeon it was easy for him to prove that in the individuals that succumb to anthrax the phagocytic reaction is very feeble, whilst in those which ultimately resist the bacillus it is very pronounced. Thiltges likewise observed that the blood and blood serum of pigeons when sown _in vitro_ with the anthrax bacillus, manifest only an insignificant bactericidal power, a fact that further warrants him in attributing great importance to phagocytosis in the maintenance of the natural immunity of the pigeon. It is remarkable that, in presence of these facts, it did not occur to the author to ask whether this fundamental difference in the mechanism of the resistance, which he thought possible in two birds so closely allied as are the pigeon and the fowl, really did exist in nature. I infer that his experiments on the fowl were made before those on the pigeon, and that the difference in his results depended specially on the fact that he had acquired greater skill in executing his later experiments.

Having observed that frogs die readily when inoculated with an anthrax bacillus that was adapted to develop at a low temperature, Dieudonné (_l.c._) endeavoured to suppress the immunity of the pigeon by using bacilli adapted to a high temperature. But the inoculation of a second generation of the anthrax bacillus, cultivated at 42° C., was borne by five pigeons without inconvenience. Even bacilli that were rendered capable, by cultivation through sixteen generations, of developing at this temperature were not in a condition to kill more than five pigeons out of thirteen inoculated. These attempts to explain immunity as due to the properties of the bacilli rather than to those of the organism of the pigeon, have therefore led to a result very different from that anticipated by Dieudonné.

[Sidenote: [157]]

The pigeon is further of interest to us because of its natural immunity against the bacillus of human tuberculosis. It resists considerable doses of this bacillus, so virulent for man and for the majority of mammals, and even for some birds (canaries and parrots). Dembinski[205], studying the mechanism of this immunity, was able to prove that the bacilli of human tuberculosis encounter in the organism of the pigeon a very great resistance from the phagocytes, especially from the macrophages. These cells fuse together around masses of bacilli and imprison them within real giant cells or polynucleated macrophages (Fig. 21). The microphages in this struggle play only a secondary part, but the resistance offered by the macrophages is a most effective one. Incapable of completely destroying the bacilli, these phagocytes exercise over them an unfavourable influence and prevent them from multiplying and exhibiting their noxious action. The importance of the defence by the macrophages comes out still more clearly when compared with what takes place if, instead of the bacillus of human tuberculosis, we inoculate into pigeons the bacillus of avian tuberculosis. In the latter case the microphages certainly promptly seize the bacilli, but being powerless against them they perish, whilst the macrophages only intervene later on and in small numbers. The result is that in the pigeon the avian bacillus becomes generalised in the organism and sets up a fatal tuberculosis.

[Illustration:

FIG. 21. Reaction of the phagocytes of the pigeon against the bacilli of human tuberculosis. ]

It must be admitted, then, that the immunity of the pigeon against the bacillus of human tuberculosis is due to the defence by the macrophages. This conclusion is corroborated by the fact that in the fowl—equally refractory against the same bacillus—there is also observed a very strong macrophagic reaction.

[Sidenote: [158]]

Nocard[206], who for several years has been carrying on studies on the relations between the bacilli of human and avian tuberculosis, conceived the idea of adapting the former to the organism of the fowl. With this object he enclosed a culture of the bacillus of human tuberculosis in a sac of collodion which he then introduced into the peritoneal cavity of fowls. Under these conditions the bacillus, protected against the aggression of phagocytes, continued to live inside the sac through whose walls the fluid part of the peritoneal lymph could diffuse. After several passages from sac to sac the human bacillus becomes acclimatised to the body of the fowl and is transformed into a variety quite comparable to the bacillus of avian tuberculosis. This experiment has definitely settled the question so long under discussion of the specific difference between the two tubercle bacilli. It has resolved it in the sense of affirming their unity; the avian bacillus is only a modified race of the same bacillus which sets up tuberculosis in man and other mammals.

In spite of the great difference between the anthrax bacillus and that of human tuberculosis, the immunity against these two bacteria, which is shown in birds, depends in every case upon the reaction of the phagocytic system.

Having rapidly glanced at natural immunity as we ascend the scale of the animal series we now come to it as it presents itself in the highest class, Mammals, a question on which it is necessary to dwell at greater length because of its great importance, and also because of the fuller study that has been given to it.

[Sidenote: [159]]

As the immunity of the Invertebrata and of the lower Vertebrata against the anthrax bacillus has furnished us with several important indications we will first endeavour to throw light on the mechanism of the resistance offered to anthrax by certain mammals. The representatives of this class being, however, for the most part extremely susceptible to this disease, examples of true natural immunity are very rare. The first place among resistant mammals is occupied by the dog. Although young dogs, as demonstrated by Strauss[207], readily take fatal anthrax, the canine species may nevertheless be regarded as possessing a real immunity, as adult dogs withstand, without inconvenience, the inoculation of large quantities of bacilli. When introduced beneath the skin these bacilli excite a local inflammation, accompanied by a very marked diapedesis of white corpuscles which at once begin to devour the bacilli. This phagocytosis has already been observed by Hess[208], Malm[209], myself, and several other investigators, so that its existence cannot be doubted. Recently, Martel[210] has demonstrated a very distinct phagocytic reaction in all those cases where he has had to deal with dogs that were refractory or not very susceptible. This reaction is shown by the ingestion of the bacteria and by the large accumulation of leucocytes at the seat of inoculation. His researches are of special interest by reason of the counter-test that he was able to make upon dogs that were susceptible to anthrax. It was demonstrated some years ago that the natural immunity of the dog against the bacillus, although very real, is, nevertheless, relative and limited. Thus Bardach[211] established the fact that dogs from whom the spleen, an organ full of phagocytes, had been removed, became susceptible to anthrax. Even dogs into whose veins he injected fine wood-charcoal powder suspended in water, with the purpose of “diverting” the phagocytosis, readily succumbed to anthrax.

Martel endeavoured to suspend the natural immunity of dogs by injecting into them phloridzin or pyrogallic acid. But he obtained much more constant results by inoculating the bacillus into rabid dogs. The organism, weakened by this terrible disease, became very susceptible to anthrax, and the rabid animal succumbed to anthrax before the rabies had completed its evolution. By its passage through the rabid dog the anthrax virus is so augmented in virulence that it becomes fatal for normal dogs. Martel succeeded also in reinforcing the bacillus isolated from a cow affected with anthrax. In all these cases where the reinforced bacilli set up a severe and rapidly fatal infection, Martel could demonstrate only a feeble phagocytic reaction.

Researches on the phagocytosis of dogs, inoculated with the anthrax bacillus, have always demonstrated a regular and constant relation between this reaction and the resistance of the organism. On the other hand, experiments undertaken for the purpose of establishing the part played by the body fluids in this immunity, have always given negative results.

[Sidenote: [160]]

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.

[Illustration:

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

[Illustration:

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

[Illustration:

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

[Illustration:

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]]

[Illustration:

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

[Illustration:

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

[Illustration:

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

[Illustration:

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

[Illustration:

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

[Illustration:

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.

[Illustration:

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

[Illustration:

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.

[Illustration:

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

[Illustration:

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]]

[Illustration:

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.

[Illustration:

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.