Immunity in infective diseases

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.).

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.

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.