Bacterial Immunity.

CHARLES J. BARTLETT, M.D., New Haven.

In response to the Committee's request for a paper upon some subject related to pathology, I shall attempt to give a brief summary of certain facts and generally accepted theories regarding immunity against bacterial infections. Much of the recent experimental work in pathology has been devoted to the study of the socalled "antibodies." These include both those formed against bacteria and their toxins, and those resulting from the introduction into the animal body of numerous other complex substances, and they are referred to as antibodies because they neutralize or destroy or in some way antagonize the substances through whose introduction they are produced. It is because our hopes for further advancement in specific serum therapy must be based upon studies of this nature that this subject has been chosen.

In order to appreciate the problems connected with the production of antibodies against bacteria, it is essential to recall the methods by which bacteria produce disease. The old theory, that this action is mechanical, has been abandoned. It now seems probable that all bacteria, which produce disease, do this by means of the toxic substances formed in their growth, and that no pathogenic bacterium is entirely wanting in this property. This toxic theory is generally accepted. These toxic substances, which are the real disease producers, do not affect all tissues alike but show a selective action. A clinical example of this is seen in the tetanus toxin, which acts upon the nervous system. Other toxins, like that of the typhoid bacillus, act chiefly upon the lympathic structures, while still others destroy red blood corpuscles. The character of the tissue acted upon by toxins is not of small importance. It may indeed be one of the chief factors in both natural and acquired immunity. Further, bacteria differ greatly in the solubility of the poisonous substances which are produced by different varieties of

the germs, and pathogenic bacteria may be divided into two groups, based upon this characteristic. The first of these forms soluble poisons. This is readily shown by cultivating these bacteria in a suitable liquid medium and then filtering this through a porcelain filter. The filtrate, freed in this way from all bacterial cells, contains the toxic products. The presence of these can be demonstrated, not by chemical tests, but by animal experimentation. These soluble poisons, resulting from bacterial growth, are known as extracellular toxins. When introduced into a susceptible animal, certain of them give rise to symptoms and lesions similar to those caused by the bacteria themselves. Only a few of the pathogenic bacteria, whose action is known, produce these extracellular toxins in large amounts. The tetanus bacillus and the dipththeria bacillus are the most conspicuous members of this group. The bacillus of bubonic plague, the cholera vibrio, the typhoid bacillus, the streptococcus and others may produce very small amounts of soluble toxins, far less than the two mentioned. They belong rather to the second group.

This second group comprises these bacteria whose chief toxic action is due to an insoluble substance. If a member of this group be grown for a short time in a liquid medium and filtered as above, the germ-free filtrate can be shown to contain little or no toxin. If, however, the bodies of these bacteria, after being killed by chloroform vapor, be injected into a susceptible animal, they can in many cases be shown to be very toxic. This toxin, whatever its nature, is evidently closely associated with the bacterial protoplasm and is not set free as a secretion by the bacterial cells. These are the so-called intracellular toxins, or endotoxins, and they are only set free by the destruction of the bacteria producing them. To this second group belong the great majority of all the pathogenic bacteria whose method of action is known, that is, they produce disease chiefly or entirely by means of intracellular toxins. In order to produce disease, they must be present in the body in considerable numbers and be destroyed. The cholera spirillum, the typhoid bacillus and pneumococcus are types of this group. The recognition of these two classes of toxins is very essential in the consideration of immunity. As will be later seen, the introduction of soluble toxins into experiment animals in proper do

sage and at suitable intervals gives rise to the productions of an antitoxin which neutralizes the action of the toxin, but in the case of the endotoxins, no such antitoxin results from their injection. An antibody is here formed to be sure, but its action is to destroy the bacteria which produce the endotoxin instead of neutralizing the toxin itself. It is an antibacterial or a bactericidal or bacteriolytic substance, not an antitoxin. The difference is a very important one from the standpoint of serum therapy.

Turning from this preliminary statement of the action of bacteria to the main purpose of the paper, the mechanism by which the animal body protects itself from bacteria and their toxins, there is found a puzzling array of experimental results. In order to get at the gist of the subject, we may disregard the protection afforded by the skin and mucous membranes, by the gastric and other digestive juices, and consider the factors at play after the bacteria or their free toxins have actually entered the body. The study of natural immunity has aided materially in throwing light upon the problem. While natural immunity is at times a matter of individual peculiarity, in its higher types it is a property of certain species of animals by which they are permanently resistant to infections to which other animals are susceptible. It is therefore evident that such natural immunity is an inherited characteristic. But even these pronounced forms of immunity frequently have their limit of resistance. If the toxic substance is introduced in very large dosage or through unusual channels, this resistance may be overcome. Thus the chicken is entirely resistant to tetanus when the toxin is introduced in the usual way, but when this is injected directly into the brain it succumbs to the disease. If, in contrast to these examples of extreme resistance to bacterial products, animals be studied whose susceptibility to specific bacterial infections is great, there is found a limit to susceptibility below which it is impossible to go. Even here in the susceptible animal, there is a certain degree of resistance though slight. Natural immunity is then in the great majority of cases a relative term, and the degree of this immunity may be modified by a variety of conditions, such as cooling the body, or in cold-blooded animals, by raising the body temperature. The cause of this natural immunity does not depend always upon any one definite factor. If living

bacteria be placed in freshly drawn normal blood, it can be demonstrated that many of them are quickly destroyed. This may be so marked that millions are killed by a small amount of blood. The same may be shown in the animal body by inoculating the blood in a loop of a vein between two ligatures. Two interesting things are to be noticed here. First, a partial preliminary destruction of bacteria occurs even though the remainder later multiply rapidly in the same blood; and, secondly, this bactericidal action is selective, not directed equally against all bacteria. If a mixture of bacteria. be taken, the blood of an animal may show much more destructive action towards one of these varieties than towards the others. Metchnikoff ascribes this power to destroy bacteria entirely to the direct or indirect action of phagocytic cells. Undoubtedly they are important agents, but recent work has shown that this action depends largely upon something in the serum which prepares the bacteria for phagocytosis. To this, reference will be made later. Further, Nuttall and other observers have shown that the normal blood serum, free from all cells, has bactericidal properties, at times as great or greater than the entire blood. Thus one of the factors in natural immunity must be accepted as a bactericidal substance in the blood. In some cases the serum of naturally immune animals contains substances which will neutralize outside of the body the toxins against which they are immune. This property may be demonstrated by mixing such serum with a fatal dose of the toxins and injecting the mixture into a susceptible animal, when the presence of the antitoxin becomes evident by the prevention of the fatal outcome. At other times neither antibacterial nor antitoxic substances can be demonstrated in the serum of naturally immune animals. As an example of this, reference may again be made to the chicken and its immunity to tetanus toxin. Though this animal possesses a high degree of immunity to tetanus toxin, its blood does not contain appreciable amounts of tetanus antitoxin. To explain immunity here, we may refer to the way in which toxin acts in the body to produce disease according to Ehrlich's theory, which has found general acceptance. He maintains that there must be a definite chemical union between the toxin and certain cells of the body for which it has an affinity. This union

of toxin and cells can only occur by means of chemical groups in the cells known as receptors, with which the toxin can combine. If none of the cells of the body have these particular atomic groups, or receptors, to anchor the toxins, the animal is immune because of this very lack of receptors. One other factor of which there is experimental evidence must be noticed, and that is the probability that cells containing these atomic groups or receptors, with which the toxin may combine, are present in very large numbers in comparatively unimportant tissues of the body, and that the fixing of the toxin by such cells, practically renders it inert. Metchnikoff has shown that the scorpion, which is absolutely immune to tetanus, stores up the tetanus toxin in the liver and retains it there for a long time. There is thus a considerable variety of recognized factors which come into play in natural immunity.

Turning from the consideration of natural immunity to that of acquired immunity, what factors give to the animal an increased resistance to the action of infectious organisms? and why may such an immunity be established against one disease, while in another it is almost entirely lacking? That there is such an immunity, frequently life long, after many of the acute infections, is a matter of general experience, but it is seen most typically and in highest degree in experiment animals. In them by repeated injections of suitable amounts of a soluble toxin, at proper intervals, a most enormous degree of toxin immunity may be established, or by repeated injection of bacterial cells, at first of slight virulence and later of exalted virulence, a similar immunity may be acquired against many varieties of bacteria. Immunity thus produced is referred to as active immunity. It is in a high degree specific, i. e., it protects only against the toxin or variety of bacteria used in producing the immunity. It is generally true that the same bacterium does not give rise to antibodies which are both highly antitoxic and antibacterial. A very few cause either the formation of an antitoxic serum or a bactericidal serum, depending upon the manipulation. The action of the antiserum is either to neutralize the toxin, or to destroy the bacteria, not both to any marked degree.