Though the causal relationship of a bacterium to a disease may be completely established by the methods given, another very important part of bacteriology is concerned with the poisons or toxins formed by bacteria.
These toxins may become free in the culture fluid, and the living bacteria may then be got rid of by filtering the fluid through a filter of unglazed porcelain, whose pores are sufficiently small to retain them. The passage of the fluid is readily effected by negative pressure produced by an ordinary water exhaust-pump. The effects of the filtrate are then tested by the methods used in pharmacology. In other instances the toxins are retained to a large extent within the bacteria, and in this case the dead bacteria are injected as a suspension in fluid. Methods have been introduced for the purpose of breaking up the bodies of bacteria and setting free the intracellular toxins. For this purpose Koch ground up tubercle bacilli in an agate mortar and treated them with distilled water until practically no deposit remained. Rowland and Macfadyen for the same purpose introduced the method of grinding the bacilli in liquid air. At this temperature the bacterial bodies are extremely brittle, and are thus readily broken up. The study of the nature of toxins requires, of course, the various methods of organic chemistry. Attempts to obtain them in an absolutely pure condition have, however, failed in important cases. So that when a "toxin" is spoken of, a mixture with other organic substances is usually implied. Or the toxin may be precipitated with other organic substances, purified to a certain extent by re-solution, re-precipitation, &c., and desiccated. A "dry toxin" is thus obtained, though still in an impure condition. Toxic substances have also been separated by corresponding methods from the bodies of those who have died of certain diseases, and the action of such substances on animals is in some cases an important point in the pathology of the disease. Another auxiliary method has been applied in this department, viz. the separation of organic substances by filtration under high pressure through a colloid membrane, gelatine supported in the pores of a porcelain filter being usually employed. It has been found, for example, that a toxin may pass through such a filter while an antitoxin may not. The methods of producing immunity are dealt with below.
[Sidenote: Bacteria as agents of disease.]
The fact that in anthrax, one of the first diseases to be fully studied, numerous bacilli are present in the blood of infected animals, gave origin to the idea that the organisms might produce their effect by using up the oxygen of the blood. Such action is now known to be quite a subsidiary matter. And although effects may sometimes be produced in a mechanical manner by bacteria plugging capillaries of important organs, _e.g._ brain and kidneys, it may now be stated as an accepted fact that all the important results of bacteria in the tissues are due to poisonous bodies or toxins formed by them. Here, just as in the general subject of fermentation, we must inquire whether the bacteria form the substances in question directly or by means of non-living ferments or enzymes. With regard to toxin formation the following general statements may be made. In certain instances, _e.g._ in the case of the tetanus and diphtheria bacilli, the production of soluble toxins can be readily demonstrated by filtering a culture in bouillon germ-free by means of a porcelain filter, and then injecting some of the filtrate into an animal. In this way the characteristic features of the disease can be reproduced. Such toxins being set free in the culture medium are often known as _extracellular_. In many cases, however, the filtrate, when injected, produces comparatively little effect, whilst toxic action is observed when the bacteria in a dead condition are used; this is the case with the organisms of tubercle, cholera, typhoid and many others. The toxins are here manifestly contained within the bodies of the bacteria, _i.e._ are _intracellular_, though they may become free on disintegration of the bacteria. The action of these intracellular toxins has in many instances nothing characteristic, but is merely in the direction of producing fever and interfering with the vital processes of the body generally, these disturbances often going on to a fatal result. In other words, the toxins of different bacteria are closely similar in their results on the body and the features of the corresponding diseases are largely regulated by the vital properties of the bacteria, their distribution in the tissues, &c. The distinction between the two varieties of toxins, though convenient, must not be pushed too far, as we know little regarding their mode of formation. Although the formation of toxins with characteristic action can be shown by the above methods, yet in some cases little or no toxic action can be demonstrated. This, for example, is the case with the anthrax bacillus; although the effect of this organism in the living body indicates the production of toxins which diffuse for a distance around the bacteria. This and similar facts have suggested that some toxins are only produced in the living body. A considerable amount of work has been done in connexion with this subject, and many observers have found that fluids taken from the living body in which the organisms have been growing, contain toxic substances, to which the name of _aggressins_ has been applied. Fluid containing these aggressins greatly increases the toxic effect of the corresponding bacteria, and may produce death at an earlier stage than ever occurs with the bacteria alone. They also appear to have in certain cases a paralysing action on the cells which act as phagocytes. The [v.03 p.0174] work on this subject is highly suggestive, and opens up new possibilities with regard to the investigation of bacterial action within the body. Not only are the general symptoms of poisoning in bacterial disease due to toxic substances, but also the tissue changes, many of them of inflammatory nature, in the neighbourhood of the bacteria. Thus, to mention examples, diphtheria toxin produces inflammatory oedema which may be followed by necrosis; dead tubercle bacilli give rise to a tubercle-like nodule, &c. Furthermore, a bacillus may give rise to more than one toxic body, either as stages in one process of change or as distinct products. Thus paralysis following diphtheria is in all probability due to a different toxin from that which causes the acute symptoms of poisoning or possibly to a modification of it sometimes formed in specially large amount. It is interesting to note that in the case of the closely analogous example of snake venoms, there may be separated from a single venom a number of toxic bodies which have a selective action on different animal tissues.
[Sidenote: Nature of toxins.]
Regarding the chemical nature of toxins less is known than regarding their physiological action. Though an enormous amount of work has been done on the subject, no important bacterial toxin has as yet been obtained in a pure condition, and, though many of them are probably of proteid nature, even this cannot be asserted with absolute certainty. Brieger, in his earlier work, found that alkaloids were formed by bacteria in a variety of conditions, and that some of them were poisonous. These alkaloids he called _ptomaines_. The methods used in the investigations were, however, open to objection, and it is now recognized that although organic bases may sometimes be formed, and may be toxic, the important toxins are not of that nature. A later research by Brieger along with Fraenkel pointed to the extracellular toxins of diphtheria, tetanus and other diseases being of proteid nature, and various other observers have arrived at a like conclusion. The general result of such research has been to show that the toxic bodies are, like proteids, precipitable by alcohol and various salts; they are soluble in water, are somewhat easily dialysable, and are relatively unstable both to light and heat. Attempts to get a pure toxin by repeated precipitation and solution have resulted in the production of a whitish amorphous powder with highly toxic properties. Such a powder gives a proteid reaction, and is no doubt largely composed of albumoses, hence the name _toxalbumoses_ has been applied. The question has, however, been raised whether the toxin is really itself a proteid, or whether it is not merely carried down with the precipitate. Brieger and Boer, by precipitation with certain salts, notably of zinc, obtained a body which was toxic but gave no reaction of any form of proteid. There is of course the possibility in this case that the toxin was a proteid, but was in so small amount that it escaped detection. These facts show the great difficulty of the problem, which is probably insoluble by present methods of analysis; the only test, in fact, for the existence of a toxin is its physiological effect. It may also be mentioned that many toxins have now been obtained by growing the particular organism in a proteid-free medium, a fact which shows that if the toxin is a proteid it may be formed synthetically by the bacterium as well as by modification of proteid already present. With regard to the nature of intracellular toxins, there is even greater difficulty in the investigation and still less is known.
Many of them, probably also of proteid nature, are much more resistant to heat; thus the intracellular toxins of the tubercle bacillus retain certain of their effects even after exposure to 100 C. Like the extracellular toxins they may be of remarkable potency; for example, fever is produced in the human subject by the injection into the blood of an extremely minute quantity of dead typhoid bacilli.
[Sidenote: Enzymes.]
We cannot as yet speak definitely with regard to the part played by enzymes in these toxic processes. Certain toxins resemble enzymes as regards their conditions of precipitation and relative instability, and the fact that in most cases a considerable period intervenes between the time of injection and the occurrence of symptoms has been adduced in support of the view that enzymes are present. In the case of diphtheria Sidney Martin obtained toxic albumoses in the spleen, which he considered were due to the digestive action of an enzyme formed by the bacillus in the membrane and absorbed into the circulation. According to this view, then, a part at least of the directly toxic substance is produced in the living body by enzymes present in the so-called toxin obtained from the bacterial culture. Recent researches go to show that enzymes play a greater part in fermentation by living ferments than was formerly supposed, and by analogy it is likely that they are also concerned in the processes of disease. But this has not been proved, and hitherto no enzyme has been separated from a pathogenic bacterium capable of forming, by digestive or other action, the toxic bodies from proteids outside the body. It is also to be noted that, as in the case of poisons of known constitution, each toxin has a minimum lethal dose which is proportionate to the weight of the animal and which can be ascertained with a fair degree of accuracy.
The action of toxins is little understood. It consists in all probability of disturbance, by means of the chemical affinities of the toxin, of the highly complicated molecules of living cells. This disturbance results in disintegration to a varying degree, and may produce changes visible on microscopic examination. In other cases such changes cannot be detected, and the only evidence of their occurrence may be the associated symptoms.
The very important work of Ehrlich on diphtheria toxin shows that in the molecule of toxin there are at least two chief atom groups--one, the "haptophorous," by which the toxin molecule is attached to the cell protoplasm; and the other the "toxophorous," which has a ferment-like action on the living molecule, producing a disturbance which results in the toxic symptoms. On this theory, susceptibility to a toxin will imply both a chemical affinity of certain tissues for the toxin molecule and also sensitiveness to its actions, and, furthermore, non-susceptibility may result from the absence of either of these two properties.
[Sidenote: Bacterial infection.]
A bacterial infection when analysed is seen to be of the nature of an intoxication. There is, however, another all-important factor concerned, viz. the multiplication of the living organisms in the tissues; this is essential to, and regulates, the supply of toxins. It is important that these two essential factors should be kept clearly in view, since the means of defence against any disease may depend upon the power either of neutralizing toxins or of killing the organisms producing them. It is to be noted that there is no fixed relation between toxin production and bacterial multiplication in the body, some of the organisms most active as toxin producers having comparatively little power of invading the tissues.
[Sidenote: The production of disease.]
We shall now consider how bacteria may behave when they have gained entrance to the body, what effects may be produced, and what circumstances may modify the disease in any particular case. The extreme instance of bacterial invasion is found in some of the septicaemias in the lower animals, _e.g._ anthrax septicaemia in guinea-pigs, pneumococcus septicaemia in rabbits. In such diseases the bacteria, when introduced into the subcutaneous tissue, rapidly gain entrance to the blood stream and multiply freely in it, and by means of their toxins cause symptoms of general poisoning. A widespread toxic action is indicated by the lesions found--cloudy swelling, which may be followed by fatty degeneration, in internal organs, capillary haemorrhages, &c. In septicaemia in the human subject, often due to streptococci, the process is similar, but the organisms are found especially in the capillaries of the internal organs and may not be detectable in the peripheral circulation during life. In another class of diseases, the organisms first produce some well-marked local lesion, from which secondary extension takes place by the lymph or blood stream to other parts of the body, where corresponding lesions are formed. In this way secondary abscesses, secondary tubercle glanders and nodules, &c., result; in typhoid fever there is secondary invasion of the mesenteric glands, and clumps of bacilli are also found in internal organs, especially the spleen, though there may be little tissue change around them. In all such cases there is seen a selective character in the distribution of the lesions, some organs being in any disease much more liable to infection than others. In still [v.03 p.0175] another class of diseases the bacteria are restricted to some particular part of the body, and the symptoms are due to toxins which are absorbed from it. Thus in cholera the bacteria are practically confined to the intestine, in diphtheria to the region of the false membrane, in tetanus to some wound.
In the last-mentioned disease even the local multiplication depends upon the presence of other bacteria, as the tetanus bacillus has practically no power of multiplying in the healthy tissues when introduced alone.
[Sidenote: Tissue changes.]
The effects produced by bacteria may be considered under the following heads: (1) tissue changes produced in the vicinity of the bacteria, either at the primary or secondary foci; (2) tissue changes produced at a distance by absorption of their toxins; (3) symptoms. The changes in the vicinity of bacteria are to be regarded partly as the _direct result_ of the action of toxins on living cells, and partly as indicating a _reaction_ on the part of the tissues. (Many such changes are usually grouped together under the heading of "inflammation" of varying degree--acute, subacute and chronic.) Degeneration and death of cells, haemorrhages, serous and fibrinous exudations, leucocyte emigration, proliferation of connective tissue and other cells, may be mentioned as some of the fundamental changes. Acute inflammation of various types, suppuration, granulation-tissue formation, &c., represent some of the complex resulting processes. The changes produced at a distance by distribution of toxins may be very manifold--cloudy swelling and fatty degeneration, serous effusions, capillary haemorrhages, various degenerations of muscle, hyaline degeneration of small blood-vessels, and, in certain chronic diseases, waxy degeneration, all of which may be widespread, are examples of the effects of toxins, rapid or slow in action. Again, in certain cases the toxin has a special affinity for certain tissues. Thus in diphtheria changes in both nerve cells and nerve fibres have been found, and in tetanus minute alterations in the nucleus and protoplasm of nerve cells.
[Sidenote: Symptoms.]
The lesions mentioned are in many instances necessarily accompanied by functional disturbances or clinical symptoms, varying according to site, and to the nature and degree of the affection. In addition, however, there occur in bacterial diseases symptoms to which the correlated structural changes have not yet been demonstrated. Amongst these the most important is fever with increased protein metabolism, attended with disturbances of the circulatory and respiratory Systems. Nervous symptoms, somnolence, coma, spasms, convulsions and paralysis are of common occurrence. All such phenomena, however, are likewise due to the disturbance of the molecular constitution of living cells. Alterations in metabolism are found to be associated with some of these, but with others no corresponding physical change can be demonstrated. The action of toxins on various glands, producing diminished or increased functional activity, has a close analogy to that of certain drugs. In short, if we place aside the outstanding exception of tumour growth, we may say that practically all the important phenomena met with in disease may be experimentally produced by the injection of bacteria or of their toxins.
[Sidenote: Susceptibility.]
The result of the entrance of a virulent bacterium into the tissues of an animal is not a disease with hard and fast characters, but varies greatly with circumstances. With regard to the subject of infection the chief factor is susceptibility; with regard to the bacterium virulence is all-important. Susceptibility, as is well recognized, varies much under natural conditions in different species, in different races of the same species, and amongst individuals of the same race. It also varies with the period of life, young subjects being more susceptible to certain diseases, _e.g._ diphtheria, than adults. Further, there is the very important factor of acquired susceptibility. It has been experimentally shown that conditions such as fatigue, starvation, exposure to cold, &c., lower the general resisting powers and increase the susceptibility to bacterial infection. So also the local powers of resistance may be lowered by injury or depressed vitality. In this way conditions formerly believed to be the causes of disease are now recognized as playing their part in predisposing to the action of the true causal agent, viz. the bacterium. In health the blood and internal tissues are bacterium-free; after death they offer a most suitable pabulum for various bacteria; but between these two extremes lie states of varying liability to infection. It is also probable that in a state of health organisms do gain entrance to the blood from time to time and are rapidly killed off. The circumstances which alter the virulence of bacteria will be referred to again in connexion with immunity, but it may be stated here that, as a general rule, the virulence of an organism towards an animal is increased by sojourn in the tissues of that animal.
The increase of virulence becomes especially marked when the organism is inoculated from animal to animal in series, the method of _passage_. This is chiefly to be regarded as an adaptation to surroundings, though the fact that the less virulent members of the bacterial species will be liable to be killed off also plays a part. Conversely, the virulence tends to diminish on cultivation on artificial media outside the body, especially in circumstances little favourable to growth.
[Sidenote: Immunity.]
By immunity is meant non-susceptibility to a given disease, or to experimental inoculation with a given bacterium or toxin. The term must be used in a relative sense, and account must always be taken of the conditions present. An animal may be readily susceptible to a disease on experimental inoculation, and yet rarely or never suffer from it naturally, because the necessary conditions of infection are not supplied in nature.
That an animal possesses natural immunity can only be shown on exposing it to such conditions, this being usually most satisfactorily done in direct experiment. Further, there are various degrees of immunity, and in this connexion conditions of local or general diminished vitality play an important part in increasing the susceptibility. Animals naturally susceptible may acquire immunity, on the one hand by successfully passing through an attack of the disease, or, on the other hand, by various methods of inoculation. Two chief varieties of artificial immunity are now generally recognized, differing chiefly according to the mode of production. In the first--_active immunity_--a reaction or series of reactions is produced in the body of the animal, usually by injections of bacteria or their products. The second--_passive immunity_--is produced by the transference of a quantity of the serum of an animal actively immunized to a fresh animal; the term is applied because there is brought into play no active change in the tissues of the second animal. The methods of active immunity have been practically applied in _preventive inoculation_ against disease; those of passive immunity have given us _serum therapeutics_. The chief facts with regard to each may now be stated.
1. _Active Immunity_.--The key to the artificial establishment of active immunity is given by the fact long established that recovery from an attack of certain infective diseases is accompanied by protection for varying periods of time against a subsequent attack. Hence follows the idea of producing a modified attack of the disease as a means of prevention--a principle which had been previously applied in inoculation against smallpox. Immunity, however, probably results from certain substances introduced into the system during the disease rather than from the disease itself; for by properly adjusted doses of the poison (in the widest sense), immunity may result without any symptoms of the disease occurring. Of the chief methods used in producing active immunity the first is by inoculation with bacteria whose virulence has been diminished, _i.e._ with an "attenuated virus." Many of the earlier methods of attenuation were devised in the case of the anthrax bacillus, an organism which is, however, somewhat exceptional as regards the relative stability of its virulence.
Many such methods consist, to speak generally, in growing the organism outside the body under somewhat unsuitable conditions, _e.g._ at higher temperatures than the optimum, in the presence of weak antiseptics, &c. The virulence of many organisms, however, becomes diminished when they are grown on the ordinary artificial media, and the diminution is sometimes accelerated by passing a current [v.03 p.0176] of air over the surface of the growth. Sometimes also the virulence of a bacterium for a particular kind of animal becomes lessened on passing it through the body of one of another species. Cultures of varying degree of virulence may be obtained by such methods, and immunity can be gradually increased by inoculation with vaccines of increasing virulence. The immunity may be made to reach a very high degree by ultimately using cultures of intensified virulence, this "supervirulent" character being usually attained by the method of _passage_ already explained. A second method is by injection of the bacterium in the dead condition, whereby immunity against the living organism may be produced. Here manifestly the dose may be easily controlled, and may be gradually increased in successive inoculations. This method has a wide application. A third method is by injections of the separated toxins of a bacterium, the resulting immunity being not only against the toxin, but, so far as present knowledge shows, also against the living organism. In the development of toxin-immunity the doses, small at first, are gradually increased in successive inoculations; or, as in the case of very active toxins, the initial injections are made with toxin modified by heat or by the addition of various chemical substances. Immunity of the same nature can be acquired in the same way against snake and scorpion poisons, and against certain vegetable toxins, _e.g._ ricin, abrin, &c.
In order that the immunity may reach a high degree, either the bacterium in a very virulent state or a large dose of toxin must ultimately be used in the injections. In such cases the immunity is, to speak generally, specific, _i.e._ applies only to the bacterium or toxin used in its production. A certain degree of non-specific immunity or increased tissue resistance may be produced locally, _e.g._ in the peritoneum, by injections of non-pathogenic organisms, peptone, nucleic acid and various other substances. In these cases the immunity is without specific character, and cannot be transferred to another animal. Lastly, in a few instances one organism has an antagonistic action to another; for example, the products of _B. pyocyaneus_ have a certain protective action against _B. anthracis_.
This method has, however, not yielded any important practical application.
2. _Passive Immunity: Anti-sera._--The development of active immunity by the above methods is essentially the result of a reactive process on the part of the cells of the body, though as yet we know little of its real nature. It is, however, also accompanied by the appearance of certain bodies in the blood serum of the animal treated, to which the name of _anti-substances_ is given, and these have been the subject of extensive study. It is by means of them that immunity (passive) can be transferred to a fresh animal. The development of anti-substances is, however, not peculiar to bacteria, but occurs also when alien cells of various kinds, proteins, ferments, &c., are injected. In fact, organic molecules can be divided into two classes according as they give rise to anti-substances or fail to do so. Amongst the latter, the vegetable poisons of known constitution, alkaloids, glucosides, &c., are to be placed. The molecules which lead to the production of anti-substances are usually known as antigens, and each antigen has a specific combining affinity for its corresponding anti-substance, fitting it as a lock does a key. The antigens, as already indicated, may occur in bacteria, cells, &c., or they may occur free in a fluid. Anti-substances may be arranged, as has been done by Ehrlich, into three main groups. In the first group, the anti-substance simply combines with the antigen, without, so far as we know, producing any change in it. The antitoxins are examples of this variety. In the second group, the anti-substance, in addition to combining with the antigen, produces some recognizable physical change in it; the precipitins and agglutinins may be mentioned as examples. In the third group, the anti-substance, after it has combined with the antigen, leads to the union of a third body called _complement_ (_alexine_ or _cytase_ of French writers), which is present in normal serum. As a result of the union of the three substances, a dissolving or digestive action is often to be observed. This is the mode of action of the anti-substances in the case of a haemolytic or bacteriolytic serum. So far as bacterial immunity is concerned, the anti-serum exerts its action either on the toxin or on the bacterium itself; that is, its action is either antitoxic or anti-bacterial. The properties of these two kinds of serum may now be considered.
[Sidenote: Antitoxic serum.]
The term "antitoxic" signifies that serum has the power of neutralizing the action of the toxin, as is shown by mixing them together outside the body and then injecting them into an animal. The antitoxic serum when injected previously to the toxin also confers immunity (passive) against it; when injected after the toxin it has within certain limits a curative action, though in this case its dose requires to be large. The antitoxic property is developed in a susceptible animal by successive and gradually increasing doses of the toxin. In the earlier experiments on smaller animals the potency of the toxin was modified for the first injections, but in preparing antitoxin for therapeutical purposes the toxin is used in its unaltered condition, the horse being the animal usually employed. The injections are made subcutaneously and afterwards intravenously; and, while the dose must be gradually increased, care must be taken that this is not done too quickly, otherwise the antitoxic power of the serum may fall and the health of the animal suffer. The serum of the animal is tested from time to time against a known amount of toxin, _i.e._ is standardized. The unit of antitoxin in Ehrlich's new standard is the amount requisite to antagonize 100 times the minimum lethal dose of a particular toxin to a guinea-pig of 250 grm. weight, the indication that the toxin has been antagonized being that a fatal result does not follow within five days after the injection. In the case of diphtheria the antitoxic power of the serum may reach 800 units per cubic centimetre, or even more. The laws of antitoxin production and action are not confined to bacterial toxins, but apply also to other vegetable and animal toxins, resembling them in constitution, viz. the vegetable toxalbumoses and the snake-venom group referred to above.
[Sidenote: Action of antitoxin.]
The production of antitoxin is one of the most striking facts of biological science, and two important questions with regard to it must next be considered, viz. how does the antitoxin act? and how is it formed within the body? Theoretically there are two possible modes of action: antitoxin may act by means of the cells of the body, _i.e._ indirectly or physiologically; or it may act directly on the toxin, _i.e._ chemically or physically. The second view may now be said to be established, and, though the question cannot be fully discussed here, the chief grounds in support of a direct action may be given. (a) The action of antitoxin on toxin, as tested by neutralization effects, takes place more quickly in concentrated than in weak solutions, and more quickly at a warm (within certain limits) than at a cold temperature. (b) Antitoxin acts more powerfully when injected along with the toxin than when injected at the same time in another part of the body; if its action were on the tissue-cells one would expect that the site of injection would be immaterial. For example, the amount necessary to neutralize five times the lethal dose being determined, twenty times that amount will neutralize a hundred times the lethal dose.
In the case of physiological antagonism of drugs this relationship does not hold. (c) It has been shown by C. J. Martin and Cherry, and by A. A.
Kanthack and Cobbett, that in certain instances the toxin can be made to pass through a gelatine membrane, whereas the antitoxin cannot, its molecules being of larger size. If, however, toxin be mixed with antitoxin for some time, it can no longer be passed through, presumably because it has become combined with the antitoxin.
Lastly it may be mentioned that when a toxin has some action which can be demonstrated in a test-tube experiment, for example, a dissolving action on red corpuscles, this action may be annulled by previously adding the antitoxin to toxin; in such a case the intervention of the living tissues is excluded. In view of the fact that antitoxin has a direct action on toxin, we may say that theoretically this may take place in one of two ways. It may produce a disintegration of the toxin molecule, or it may combine with it to produce a body whose combining affinities are satisfied.
The latter view, first advocated by [v.03 p.0177] Ehrlich, harmonizes with the facts established with regard to toxic action and the behaviour of antitoxins, and may now be regarded as established. His view as to the dual composition of the toxin molecule has already been mentioned, and it is evident that if the haptophorous or combining group has its affinity satisfied by union with antitoxin, the toxin will no longer combine with living cells, and will thus be rendered harmless. One other important fact in support of what has been stated is that a toxin may have its toxic action diminished, and may still require the same amount of antitoxin as previously for neutralization. This is readily intelligible on the supposition that the toxophorous group is more labile than the haptophorous. There is, however, still dispute with regard to the exact nature of the union of toxin and antitoxin. Ehrlich's view is that the two substances form a firm combination like a strong acid and a base. He found, however, that if he took the largest amount of toxin which was just neutralized by a given amount of antitoxin, much more than a single dose of toxin had to be added before a single dose was left free. For example, if 100 doses of toxin were neutralized by a unit of antitoxin (_v. supra_) it might be that 125 doses would need to be added to the same amount of antitoxin before the mixture produced a fatal result when it was injected.
This result, which is usually known now as the "Ehrlich phenomenon," was explained by him on the supposition that the "toxin" does not represent molecules which are all the same, but contains molecules of different degrees of combining affinity and of toxic action. Accordingly, the most actively toxic molecules will be neutralized first, and those which are left over, that is, uncombined with antitoxin, will have a weaker toxic action. This view has been assailed by Thorvald Madsen and S. A. Arrhenius, who hold that the union of toxin and antitoxin is comparatively loose, and belongs to the class of reversible actions, being comparable in fact with the union of a weak acid and base. If such were the condition there would always be a certain amount both of free toxin and of free antitoxin in the mixture, and in this case also considerably more than a dose of toxin would have to be added to a "neutral mixture" before the amount of free toxin was increased by a dose, that is, before the mixture became lethal. It may be stated that while in certain instances the union of toxin and antitoxin may be reversible, all the facts established cannot be explained on this simple hypothesis of reversible action. Still another view, advocated by Bordet, is that the union of toxin and antitoxin is rather of physical than of strictly chemical nature, and represents an interaction of colloidal substances, a sort of molecular deposition by which the smaller toxin molecule becomes entangled in the larger molecule of antitoxin. Sufficient has been said to show that the subject is one of great intricacy, and no simple statement with regard to it is as yet possible. We are probably safe in saying, however, that the molecules of a toxin are not identical but vary in the degree of their combining affinities, and also in their toxic action, and that, while in some cases the combination of anti-substances has been shown to be reversible, we are far from being able to say that this is a general law.
[Sidenote: Formation of antitoxin.]
The origin of antitoxin is of course merely a part of the general question regarding the production of anti-substances in general, as these all combine in the same way with their homologous substances and have the same character of specificity. As, however, most of the work has been done with regard to antitoxin production we may consider here the theoretical aspect of the subject. There are three chief possibilities: (a) that the antitoxin is a modification of the toxin; (b) that it is a substance normally present, but produced in excess under stimulation of the toxin; (c) that it is an entirely new product. The first of these, which would imply a process of a very remarkable nature, is disproved by what is observed after bleeding an animal whose blood contains antitoxin. In such a case it has been shown that, without the introduction of fresh toxin, new antitoxin appears, and therefore must be produced by the living tissues. The second theory is the more probable _a priori_, and if established removes the necessity for the third. It is strongly supported by Ehrlich, who, in his so-called "side-chain" (_Seitenkette_) theory, explains antitoxin production as an instance of regeneration after loss. Living protoplasm, or in other words a biogen molecule, is regarded as consisting of a central atom group (_Leistungskern_), related to which are numerous secondary atom groups or side-chains, with unsatisfied chemical affinities. [Sidenote: "Side-chain" theory.] The side-chains constitute the means by which other molecules are added to the living molecule, _e.g._ in the process of nutrition. It is by means of such side-chains that toxin molecules are attached to the protoplasm, so that the living molecules are brought under the action of the toxophorous groups of the toxins. In antitoxin production this combination takes place, though not in sufficient amount to produce serious toxic symptoms. It is further supposed that the combination being of somewhat firm character, the side-chains thus combined are lost for the purposes of the cell and are therefore thrown off. By the introduction of fresh toxin the process is repeated and the regeneration of side-chains is increased. Ultimately the regeneration becomes an over-regeneration and free side-chains produced in excess are set free and appear in the blood as antitoxin molecules. In other words the substances, which when forming part of the cells fix the toxin to the cells, constitute antitoxin molecules when free in the serum. This theory, though not yet established, certainly affords the most satisfactory explanation at present available. In support of it there is the remarkable fact, discovered by A. Wassermann and Takaki in the case of tetanus, that there do exist in the nervous system molecules with combining affinity for the tetanus toxin. If, for example, the brain and spinal cord removed from an animal be bruised and brought into contact with tetanus toxin, a certain amount of the toxicity disappears, as shown by injecting the mixture into another animal. Further, these molecules in the nervous system present the same susceptibility to heat and other physical agencies as does tetanus antitoxin. There is therefore strong evidence that antitoxin molecules do exist as part of the living substance of nerve cells. It has, moreover, been found that the serum of various animals has a certain amount of antitoxic action, and thus the basis for antitoxin production, according to Ehrlich's theory, is afforded. The theory also supplies the explanation of the power which an animal possesses of producing various antitoxins, since this depends ultimately upon susceptibility to toxic action. The explanation is thus carried back to the complicated constitution of biogen molecules in various living cells of the body. It may be added that in the case of all the other kinds of anti-substances, which are produced by a corresponding reaction, we have examples of the existence of traces of them in the blood serum under normal conditions. We are, accordingly, justified in definitely concluding that their appearance in large amount in the blood, as the result of active immunization, represents an increased production of molecules which are already present in the body, either in a free condition in its fluids or as constituent elements of its cells.
[Sidenote: Anti-bacterial serum.]
In preparing anti-bacterial sera the lines of procedure correspond to those followed in the case of antitoxins, but the bacteria themselves in the living or dead condition or their maceration products are always used in the injections. Sometimes dead bacteria, living virulent bacteria, and living supervirulent bacteria, are used in succession, the object being to arrive ultimately at a high dosage, though the details vary in different instances. The serum of an animal thus actively immunized has powerful protective properties towards another animal, the amount necessary for protection being sometimes almost inconceivably small. As a rule it has no action on the corresponding toxin, _i.e._ is not antitoxic. In addition to the protective action, such a serum may possess activities which can be demonstrated outside the body. Of these the most important are (a) bacteriolytic or lysogenic action, (b) agglutinative action, and (c) opsonic action.
[Sidenote: (a) Lysogenic action.]
The first of these, lysogenic or bacteriolytic action, consists in [v.03 p.0178] the production of a change in the corresponding bacterium whereby it becomes granular, swells up and ultimately may undergo dissolution.
Pfeiffer was the first to show that this occurred when the bacterium was injected into the peritoneal cavity of the animal immunized against it, and also when a little of the serum of such an animal was injected with the bacterium into the peritoneum of a fresh, _i.e._ non-immunized animal.
Metchnikoff and Bordet subsequently devised means by which a similar change could be produced _in vitro_, and analysed the conditions necessary for its occurrence. It has been completely established that in this phenomenon of lysogenesis there are two substances concerned, one specially developed or developed in excess, and the other present in normal serum. The former (_Immunkorper_ of Ehrlich, _substance sensibilisatrice_ of Bordet) is the more stable, resisting a temperature of 60 C., and though giving the specific character to the reaction cannot act alone. The latter is ferment-like and much more labile than the former, being readily destroyed at 60 C. It may be added that the protective power is not lost by exposure to the temperature mentioned, this apparently depending upon a specific anti-substance. Furthermore, lysogenic action is not confined to the case of bacteria but obtains also with other organized structures, _e.g._ red corpuscles (Bordet, Ehrlich and Morgenroth), leucocytes and spermatozoa (Metchnikoff). That is to say, if an animal be treated with injections of these bodies, its serum acquires the power of dissolving or of producing some disintegrative effect in them. The development of the immune body with specific combining affinity thus presents an analogy to antitoxin production, the difference being that in lysogenesis another substance is necessary to complete the process. It can be shown that in many cases when bacteria are injected the serum of the treated animal has no bacteriolytic effect, and still an immune body is present, which leads to the fixation of complement; in this case bacteriolysis does not occur, because the organism is not susceptible to the action of the complement. In all cases the important action is the binding of complement to the bacterium by means of the corresponding immune body; whether or not death of the bacterium occurs, will depend upon its susceptibility to the action of the particular complement, the latter acting like a toxin or digestive ferment. It is to be noted that in the process of immunization complement does not increase in amount; accordingly the immune serum comes to contain immune body much in excess of the amount of complement necessary to complete its action. An important point with regard to the therapeutic application of an anti-bacterial serum, is that when the serum is kept _in vitro_ the complement rapidly disappears, and accordingly the complement necessary for the production of the bactericidal action must be supplied by the blood of the patient treated. This latter complement may not suit the immune body, that is, may not be fixed to the bacterium by means of it, or if the latter event does occur, may fail to bring about the death of the bacteria. These circumstances serve, in part at least, to explain the fact that the success attending the use of anti-bacterial sera has been much inferior to that in the case of antitoxic sera.
[Sidenote: (b) Agglutination.]
Another property which may be possessed by an anti-bacterial serum is that of agglutination. By this is meant the aggregation into clumps of the bacteria uniformly distributed in an indifferent fluid; if the bacterium is motile its movement is arrested during the process. The process is of course observed by means of the microscope, but the clumps soon settle in the fluid and ultimately form a sediment, leaving the upper part clear.
This change, visible to the naked eye, is called _sedimentation_. B. J. A.
Charrin and G. E. H. Roger first showed in the case of _B. pyocyaneus_ that when a small quantity of the homologous serum (_i.e._ the serum of an animal immunized against the bacterium) was added to a fluid culture of this bacillus, growth formed a sediment instead of a uniform turbidity.
Gruber and Durham showed that sedimentation occurred when a small quantity of the homologous serum was added to an emulsion of the bacterium in a small test-tube, and found that this obtained in all cases where Pfeiffer's lysogenic action could be demonstrated. Shortly afterwards Widal and also Grunbaum showed that the serum of patients suffering from typhoid fever, even at an early stage of the disease, agglutinated the typhoid bacillus--a fact which laid the foundation of serum diagnosis. A similar phenomenon has been demonstrated in the case of Malta fever, cholera, plague, infection with _B. coli_, "meat-poisoning" due to Gartner's bacillus, and various other infections. As regards the mode of action of agglutinins, Gruber and Durham considered that it consists in a change in the envelopes of the bacteria, by which they swell up and become adhesive. The view has various facts in its support, but F. Kruse and C. Nicolle have found that if a bacterial culture be filtered germ-free, an agglutinating serum still produces some change in it, so that particles suspended in it become gathered into clumps. E. Duclaux, for this reason, considers that agglutinins are coagulative ferments.
The phenomenon of agglutination depends essentially on the union of molecules in the bacteria--the agglutinogens--with the corresponding agglutinins, but another essential is the presence of a certain amount of salts in the fluid, as it can be shown that when agglutinated masses of bacteria are washed salt-free the clumps become resolved. The fact that agglutinins appear in the body at an early stage in a disease has been taken by some observers as indicating that they have nothing to do with immunity, their development being spoken of as a reaction of infection.
This conclusion is not justified, as we must suppose that the process of immunization begins to be developed at an early period in the disease, that it gradually increases, and ultimately results in cure. It should also be stated that agglutinins are used up in the process of agglutination, apparently combining with some element of the bacterial structure. In view of all the facts it must be admitted that the agglutinins and immune bodies are the result of corresponding reactive processes, and are probably related to one another. The development of all antagonistic substances which confer the special character on antimicrobic sera, as well as antitoxins, may be expressed as the formation of bodies with specific combining affinity for the organic substance introduced into the system--toxin, bacterium, red corpuscle, &c., as the case may be. The bacterium, being a complex organic substance, may thus give rise to more than one antagonistic or combining substance.
[Sidenote: (c) Opsonic action.]
By opsonic action is meant the effect which a serum has on bacteria in making them more susceptible to phagocytosis by the white corpuscles of the blood (_q.v._). Such an effect may be demonstrated outside the body by making a suitable mixture of (a) a suspension of the particular bacterium, (b) the serum to be tested, and (c) leucocytes of a normal animal or person. The mixture is placed in a thin capillary tube and incubated at 37 C. for half an hour; a film preparation is then made from it on a glass slide, stained by a suitable method and then examined microscopically. The number of bacteria contained within a number of, say fifty, leucocytes can be counted and the average taken. In estimating the opsonic power of the serum in cases of disease a control with normal serum is made at the same time and under precisely the same conditions. The average number of bacteria contained within leucocytes in the case tested, divided by the number given by the normal serum, is called the _phagocytic index_. Wright and Douglas showed that under these conditions phagocytosis might occur when a small quantity of normal serum was present, whereas it was absent when normal salt solution was substituted for the serum; the latter thus contained substances which made the organisms susceptible to the action of the phagocytosis. They further showed that this substance acted by combining with the organisms and apparently producing some alteration in them; on the other hand it had no direct action on the leucocytes. This opsonin of normal serum is very labile, being rapidly destroyed at 55 C.; that is, a serum heated at this temperature has practically no greater effect in aiding phagocytosis than normal salt solution has. Various observers had previously found that the serum of an animal immunized against [v.03 p.0179] a particular bacterium had a special action in bringing about phagocytosis of that organism, and it had been found that this property was retained when the serum was heated at 55 C. It is now generally admitted that at least two distinct classes of substances are concerned in opsonic action, that thermostable immune opsonins are developed as a result of active immunization and these possess the specific properties of anti-substances in general, that is, act only on the corresponding bacterium. On the contrary the labile opsonins of normal serum have a comparatively general action on different organisms. It is quite evident that the specific immune-opsonins may play a very important part in the phenomena of immunity, as by their means the organisms are taken up more actively by the phagocytic cells, and thereafter may undergo rapid disintegration.
The opsonic action of the serum has been employed by Sir A. Wright and his co-workers to control the treatment of bacterial infections by vaccines; that is, by injections of varying amounts of a dead culture of the corresponding bacterium. The object in such treatment is to raise the opsonic index of the serum, this being taken as an indication of increased immunity. The effect of the injection of a small quantity of vaccine is usually to produce an increase in the opsonic index within a few days. If then an additional quantity of vaccine be injected there occurs a fall in the opsonic index (negative phase) which, however, is followed later by a rise to a higher level than before. If the amounts of vaccine used and the times of the injection are suitably chosen, there may thus be produced by a series of steps a rise of the opsonic index to a high level. One of the chief objects in registering the opsonic power in such cases is to avoid the introduction of additional vaccine when the opsonic index is low, that is, during the negative phase, as if this were done a further diminution of the opsonic action might result. The principle in such treatment by means of vaccines is to stimulate the general production of anti-substances throughout the body, so that these may be carried to the sites of bacterial growth, and aid the destruction of the organisms by means of the cells of the tissues. A large number of favourable results obtained by such treatment controlled by the observation of the opsonic index have already been published, but it would be unwise at present to offer a decided opinion as to the ultimate value of the method.
Active immunity has thus been shown to be associated with the presence of certain anti-substances in the serum. After these substances have disappeared, however, as they always do in the course of time, the animal still possesses immunity for a varying period. This apparently depends upon some alteration in the cells of the body, but its exact nature is not known.