Before quitting the group of bacteria we must cast a glance at the mechanism of acquired immunity against representatives of the group of spherical micro-organisms. Amongst the cocci the streptococci have been especially studied as regards this immunity. For long great difficulties were encountered in vaccinating animals against these chain cocci, but Roger[353], Marmorek[354], Denys and Leclef[355] overcame these obstacles and succeeded in immunising the rabbit, one of the most susceptible species, to their pathogenic action. More recently the larger mammals, notably the horse, have been successfully immunised. A certain number of important facts, the knowledge of which is useful to complete the survey of the phenomena of acquired immunity, have thus been collected.
Roger set himself to study the properties of the blood serum of rabbits vaccinated against the streptococcus, and established the fact that this fluid had not the slightest appreciable bactericidal action; the streptococcus grew in it just as well as in the serum of fresh unvaccinated rabbits. When, however, he injected cultures grown in the serum of immunised animals into rabbits, these rabbits did not die and presented only transient and insignificant lesions. From this fact Roger concluded that there must be an attenuation of the streptococcus by the immune serum, a view which was shared by several other observers. In formulating this view, however, he had not taken into account the possibility that this serum acted not upon the coccus that had developed in it but upon the organism of the animal into which it was injected. Bordet[356], indeed, was able to show that the streptococcus which grows in the serum of immunised animals is in no way weakened in virulence. When he took a race very virulent for the rabbit (Marmorek’s streptococcus) and injected a minimal dose of a culture grown in the serum of immunised animals, the rabbits died just as did the control animals, because the amount of serum introduced was too small to exert any influence. So also, when he filtered this culture and got rid of the serum bathing the streptococci, it was found to be just as virulent as that grown in the serum of susceptible unvaccinated animals.
In confirmation of the discovery made by Roger with the serum of vaccinated rabbits, Bordet showed that the blood serum of horses highly immunised against the streptococcus did not exhibit any bactericidal action. Moreover, he found that this serum caused the development of somewhat agglutinated streptococci and that it was capable of throwing streptococci grown on the ordinary media into clumps. Summing up his researches on the properties of this serum Bordet concludes that it “causes no profound change in the streptococcus. The vegetative character of the coccus is not appreciably diminished, and its morphology remains the same save for certain variations in the length of the chains. Even the agglutinative power, recognised in numerous serums by recent researches, is, in the antistreptococcic serum, developed but slightly” (p. 196).
More recently von Lingelsheim[357] has studied the properties of the serum of animals which he had thoroughly vaccinated against the streptococcus. He observed a certain slowing of the development of the coccus in this serum as compared with the growth in cultures made in the serum of normal, susceptible animals. But this retardation was slight and transient, and exhibited itself especially in serums to which von Lingelsheim, following Denys, had added leucocytes.
Von Lingelsheim also noted a certain degree of agglutination of the streptococcus by the serum of vaccinated animals, although this was much more feeble than in the case of the cholera vibrio or the typhoid bacillus, when agglutinated by their corresponding serums. Speaking generally, he regarded the direct action of the body fluids as insufficient to bring about the rapid destruction of the streptococci in the vaccinated organism. “Since the action of the bactericidal substances is limited in time, the streptococci are able to adapt themselves to these substances and recover their former energy. As the phenomena of extracellular solution, of such a form as those observed under the influence of the cholera antibodies, are absent in the case of the streptococcus and as, on the other hand, a considerable ingestion of these organisms by the leucocytes is observed ... we must seek in the activity of these cells a second important element of the defence of the animal organism” (p. 78).
To Salimbeni[358], who has carried out in my laboratory an investigation on this subject, we are indebted for the most reliable information on the phagocytic reaction in acquired immunity against the streptococcus. He studied specially the phenomena in the subcutaneous tissue of a horse, hypervaccinated against Marmorek’s streptococcus; this animal received in all, at several injections, about five litres of living culture. In spite of this refractory condition, an oedema at the point of inoculation was soon produced; in this the micro-organisms remained free and the leucocytes were sparse. But the cellular reaction, at first insignificant, developed with great rapidity and many leucocytes, amongst which the macrophages were much the more numerous, were attracted. The phagocytosis was still delayed for some time, but it continued to increase and 20 to 24 hours after the inoculation it was complete. As soon as the phagocytosis was well established the oedema began to disappear. In the thick exudation, containing a mass of leucocytes, the macrophages are filled with a very large number of streptococci packed together. These cocci develop inside the cells, cause them to burst and again become free. A fresh arrival of leucocytes, however, takes place, this time mainly microphages. These microphages seize the free streptococci that have struggled so victoriously against the macrophages; this second phagocytic phase is final. The streptococci still remain alive inside the microphages for some days, but ultimately are killed and digested by the phagocytes. At a period when, 5 or 6 days after injection, insignificant or isolated traces of streptococci are to be found in the microphages, the exudation when sown in nutritive media still gives abundant cultures. The incidents of this struggle between the streptococcus and the animal organism demonstrate the important part played by the phagocytes. The fact that the macrophages perish and allow the cocci to escape, clearly proves that these cocci have been ingested alive and virulent, and consequently that the fluid of the exudation was incapable of destroying or even of attenuating them. The macrophages, also, were powerless to bring about this result and the intervention of the microphages was necessary to cause the disappearance of the cocci. It is, however, always the phagocytes which ensure the final resistance of the animal.
In presence of these very precise results obtained from the work of Salimbeni, a work which I followed very closely, the previous researches by Denys and Leclef (l.c.) made under less favourable conditions on vaccinated rabbits are deprived of their importance. These observers wished to get an idea of the difference between the reactions of the animal organism (a) after the injection of streptococci into the pleural cavity of immunised rabbits, and (b) after injection into that of normal susceptible rabbits. They killed the inoculated animals and found a very marked diminution of micro-organisms in the pleuritic exudation of the former. This diminution could not be attributed to a lysis of the streptococci by the body fluids, because there were never any signs of such destruction. Nor could the phagocytosis, very feeble at first, be considered as the cause of the disappearance of a large number of the streptococci. Denys and Leclef put forward a third hypothesis, which attributed this disappearance to the rapid resorption by the lymph stream of the injected fluid containing the organisms. Going over the record of their experiments it will be seen that in vaccinated rabbits the quantity of pleuritic exudation was always very much less than in normal rabbits. In presence of this feature there is reason to ask whether, in the case of the streptococci, a large number of these organisms were not fixed, along with the leucocytes, on the walls of the pleura, as in guinea-pigs that are inoculated intraperitoneally? Instead of being satisfied with merely examining the fluid exudation, the surface of the pleura should have been scraped in order to ascertain whether the phagocytic reaction was localised in this region.
In any case such incomplete results on the active immunity of rabbits in no way weaken the positive results obtained in the subcutaneous tissue of the horse, in which the phagocytic reaction plays a really preponderant part.
This example of the streptococci completes our series of bacteria in which we have studied their relations with the properties of the animal organism that has acquired immunity. We have still to see whether the acquired immunity against micro-organisms of animal origin is subject to the same law as that against bacteria.
For some years past a zealous study of the infectious diseases produced by animal micro-organisms has been carried out. Besides malaria, which occupies a most important position, attention has been directed to certain diseases in domestic animals that are set up by endoglobular haematozoa and by flagellata, and a fairly large number of accurate data have been collected with regard to Texas fever and its parasite the Piroplasma bigeminum, as well as upon the epizootic diseases due to Trypanosomata (Tsetse fly disease or Nagana, “Dourine,” etc.).
We are indebted to Smith and Kilborne[359] for the earliest information concerning the acquired immunity of Bovidae against Texas fever. R. Koch[360] then added some very precise observations on the immunity of calves which had been inoculated with parasites attenuated in the body of the tick (Boophilus bovis). Lignières[361], who devoted much attention to this question in the Argentine Republic, has discovered a sure method of vaccinating the Bovidae against the “Tristeza,” the local name for Texas fever. He brought to Alfort specimens of attenuated haematozoa, and in Nocard’s presence performed successful vaccination experiments. Lignières is now engaged in devising a practical method of ensuring immunity under the special conditions found in the home of the “Tristeza.” Up to the present, however, there are no sufficient data as to the mechanism of the acquired immunity in this case. We have fuller information as to the essential phenomena observed in the organism of the rat vaccinated against Trypanosoma lewisi. We owe to Mme. L. Rabinowitsch and Dr Kempner[362] the first important data as to the possibility of immunising white or piebald rats against the disease produced by the flagellated infusorian. They noted that these animals when inoculated with the blood of grey rats containing Trypanosomata acquire a very transitory disease which, however, confers an immunity against any subsequent infection. The flagellated organisms disappear from the blood within a few weeks, after which fresh injections of these parasites have no pathogenic effect.
Laveran and Mesnil[363] confirmed these observations, and in addition made careful observations on the mechanism of this acquired immunity. After making several inoculations with blood containing Trypanosomata into white rats, they made a study of the properties of the blood serum of these immunised animals. First they established the fact that this serum exerts no microbicidal action on the Trypanosomata, but it agglutinates them without, however, rendering them motionless:—“The masses may be resolved into rosettes in which the Trypanosomata, united merely by their posterior extremities, have their flagella free and motile at the periphery.”
Laveran and Mesnil then studied the phenomena evolved in the refractory organism. When injected into the peritoneal cavity of immunised rats the Trypanosomata are not acted upon injuriously by the body fluids. They are, however, devoured by the leucocytes. Laveran and Mesnil thus express themselves on this subject: “... we have demonstrated clearly and repeatedly that the Trypanosomata are ingested alive, perfectly isolated and very motile, by phagocytes, and we have followed the details of this process of ingestion which recalls that of the ingestion of spirilla by the leucocytes of the guinea-pig. We consider, therefore, that the immunity is phagocytic in character.”
The main facts on acquired immunity established in connection with the most diverse micro-organisms, facts just described, may already be said to lead to certain general conclusions. They indicate in the first place that acquired immunity is accompanied by phenomena more complicated than those observed in natural immunity. In the two categories of processes observed in acquired immunity the phagocytic reaction is the only one that can be said to be constant. We find it in those examples in which the influence of the fluids of the body is most manifest, as in the experimental cholera peritonitis of the guinea-pig, as well as in those cases where the humoral action is most feeble, as in anthrax or in the Trypanosoma disease of rats. We have, however, still to establish the relations that exist between phagocytosis and the part played by the fluids of the immunised animal, in order that we may, as far as possible, present a general picture of the inner mechanism of acquired immunity against micro-organisms. To attain this result we must place the reader in possession of further well-established facts, and we must postpone its discussion to the following chapter, which will be entirely devoted to the above-mentioned problem.