Volume 3, Part 1, Slice 2 Part 5 (1/2)

Though the causal relations.h.i.+p 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 pa.s.sage 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 pa.s.s through such a filter while an ant.i.toxin 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 teta.n.u.s 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 a.n.a.logous 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 a.s.serted 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, teta.n.u.s 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 alb.u.moses, hence the name _toxalb.u.moses_ 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 a.n.a.lysis; 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 quant.i.ty 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 alb.u.moses 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 a.n.a.logy 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 const.i.tution, 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 a.s.sociated 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 a.n.a.lysed 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 circ.u.mstances 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 cla.s.s 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 cla.s.s 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 teta.n.u.s to some wound.

In the last-mentioned disease even the local multiplication depends upon the presence of other bacteria, as the teta.n.u.s 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 teta.n.u.s 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 const.i.tution of living cells. Alterations in metabolism are found to be a.s.sociated 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 a.n.a.logy 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 circ.u.mstances. 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 circ.u.mstances 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 _pa.s.sage_. 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 circ.u.mstances 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 pa.s.sing 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--_pa.s.sive immunity_--is produced by the transference of a quant.i.ty 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 pa.s.sive 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 pa.s.sing 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 pa.s.sing 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 _pa.s.sage_ 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.