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.

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.

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.

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.

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.

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.

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.

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.

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.