There is but one way of characterizing the toxic poisons secreted by microbes, and that is to apply to them the name of the microbes generating them; thus the soluble and toxic poison of the tetanus bacilli has received the nametetanus toxin.
In toxic microbial cultures it is necessary to distinguish the toxins proper from the toxic alkaloids (ptomaines) which generally accompany them; this is easily accomplished by evaporating the solution in a vacuum at about 30°C., and then treating with alcohol and ether, in which the alkaloids are soluble, while the true toxins are insoluble. By fractional precipitation with alcohol it is easy to isolate the peptones and true toxins.
The microbial toxins possess two essential properties; one the pyogenic property, thanks to which the toxins first attract, then destroy the white blood corpuscles or leucocytes, and transform them into pus, and the other the pyretogenic property, which appears to belong only quiteindirectly to the pyogenic substance. The toxins in general retard the heart action.
We will not speak of the distinctions it has been sought to establish between the substances which possess these different properties, but will at once take up the discussion of several of the microbial toxins.
Anthrax Toxin100(from Bacillus Anthracis).—We will describe the preparation of this toxin as a type.
The cultures of the bacillus are made in Liebig's bouillon, to which has been added 0.1% of fibrin, the whole being carefully sterilized for a long time at 110° C. The cultures medium is inoculated with a drop of blood taken from the heart or spleen of an animal that has died of anthrax. At the end of a week, the culture is filtered, and the filtrate acidulated with a little acetic acid and precipitated by adding powdered ammonium sulphate. The flocculent precipitate is collected, washed, dissolved in distilled water, and dialyzed. The dialyzed solution is concentrated in vacuo at 40-45° C., and precipitated by adding to it alcohol. The precipitate formed is then collected and dried.101
In this manner there is obtained a grayish-white substance which is soluble in water, and which is fatal in large doses, but which, given in repeated small doses, confers immunity against anthrax.
According to Hankin, it seems that the toxic property of this toxin is due to an albumose.
Marchoux102has been able to confer immunity upon sheep by injecting first small quantities of the filtered culture of the anthrax bacilli, and then the virulent anthrax itself.
The animals thus rendered immune yield a serum which may be used as a vaccin against anthrax, and which even possesses curative properties under certain conditions.
In every case the acquired immunity is only temporary. We will recall to recollection the method employed by Pasteur for vaccinating against anthrax, using attenuated cultures, a method which is practiced daily at the present time.103
From the cultures of symptomatic anthrax (Bacillus Chauvæ) Chauvée extracted a very active toxin which can withstand without impairment a temperature of 110°C.104Roux105has shown thatthe serum of animals that have succumbed to the symptomatic anthrax is capable of vaccinating against this disease; we have here a new proof that the antitoxin is in fact a product of the defense of the cells of the organism, and the author mentioned has been able to vaccinate guinea-pigs by injecting into the peritoneum culture bouillon sterilized by heating to 115° C. or by filtering through porcelain.
Tubercular Toxin.—The culture bouillons of Koch's bacillus contain one or more active substances which constitute, and which is at the present designated as, tuberculin.106Koch's therapeutic tuberculin is obtained by evaporating to one-tenth its volume a culture bouillon of Koch's tubercle bacilli prepared from a 4-per cent. glycerinic mutton bouillon, and filtering through porcelain. By fractional precipitation it is possible to obtain from the crude tuberculin so prepared a product which is considered as pure tuberculin, and which possesses considerable activity.
Prolonged boiling on the water-bath completely destroys the activity of this tuberculin, which moreover hardly ever keeps longer than three weeks. It has been found possible to preserve it for an indefinite period, however, by adding toit 30 to 40 per cent. of glycerin. It possesses all the general reactions of albuminoids.
Tuberculin is not toxic in the proper sense of the word. Injected in small quantities into the healthy human being107and into healthy animals, it exerts no effect; on the other hand, however, in tubercular organisms, even in incipient stages of the disease, even where it is almost impossible to make a clinical diagnosis, the injection of very small quantities develops a lively and characteristic reaction.108
Grasset and Vedel consider the tuberculin as an excellent means of diagnosing tuberculosis in man, but in such a case it is necessary to operate with the greatest caution, with very small quantities of the tuberculin, and to feel, in some sort, the sensitiveness of the patient, particularly in the case of children.
It is chiefly for the diagnosis of tuberculosis in cattle, however, that tuberculin is valuable. Thanks to Nocard, the procedure has to-day become a common practice. The injection of a fairly large dose, 0.3 to 0.4 Gm., according to the size of the animal, causes, in about ten hours or so, if the animal is tuberculous, a strong febrile reaction with an elevation of temperature of 1.5 to 3° C., whereas if the animal is not tuberculous no such reaction takes place.
Cases in which tuberculosis is far advanced, and in which the organism is impregnated with tuberculin, do not react after the injection of tuberculin.109
Tuberculin does not confer immunity, and the bacillus retains all its virulence, even in injected tissues; nevertheless, the return to health of animals in which injections have been recently made may be due to the action of large doses of the serum; and on the other hand the tuberculin, in large quantities, may render the location unsuitable for the development of the tubercle bacilli.
Diphtheria Toxin.—The most characteristic property of the diphtheria bacillus is the production, in culture media, of a special toxic substance which has been nameddiphtheritic toxin; this name, however, has come to be also extended to a liquid in which the bacilli have lived, and which has been sterilized by filtration or by any other suitable process.
Roux and Yersin110were the first to affirm that diphtheria is an autointoxication caused by a very active poison formed by the microbe in the restricted locality where it develops. In order to obtain thistoxin111a culture of the bacillus is first made in a mutton bouillon made strongly alkaline with sodium carbonate (10 grams per liter), and with the addition of 2 per cent. of peptone. At the end of about one month, the culture being kept at about 37° C., the liquid is filtered through porcelain. It is indispensable to employ a very virulent bacillus; it is hence frequently advantageous to increase the virulence and toxigenic power of the bacilli it is desired to use.
The toxic liquid obtained is exceedingly powerful: 0.1 Cc. kills a rabbit in forty-eight hours. This toxin is very sensitive to the effects of heat. When heated to 65° C. it loses almost all its toxicity; at 70º C. it becomes innocuous; and it only requires to be heated to 100° C. for fifteen minutes in order to lose all immediate activity even in large doses. Nevertheless toxins thus weakened are capable of proving fatal to an animal even after five or six months.
Light, oxygen, ozone and all oxidizers destroy the active principle of the diphtheria toxin, which is, moreover, rendered almost inactive by organic acids.
This toxin is capable of diffusing through animal membranes, a fact that is in agreement with the toxic effect seen in a subject attacked with diphtheria, and due to the toxin passing through the mucosa. In spite of this property, however, the diphtheritic poison may be taken into the stomach without any pernicious results.
Roux and Yersin have shown that, like all the diastases, it may be precipitated from its solutions by the development, within these, of certain precipitates, particularly calcium phosphate. It is precipitated from its solutions by alcohol, as has been observed also in the case of diastatic solutions. All the toxic substance is contained in the albuminous precipitate thus obtained; but the prolonged action of alcohol, or repeated successive precipitations, alter it finally. Diphtheria toxin is likewise precipitated by the reagents for albumoses, particularly sodium sulphate in saturated solution. This procedure has been utilized by Brieger and Fraenkel for preparing the pure toxin, which they obtained in the form of very light, brilliant white, amorphous flocks, affording all the principal reactions of the soluble albumoses (biuret, xanthoproteic, Millon's), and which they characterized as a toxalbumin.
On injecting into healthy animals this diphtheria toxin attenuated by sufficiently heating at 70° C, employing at first small doses, and gradually increasing, it is possible to immunize them against diphtheria, as was first demonstrated by Carl Fraenkel.
Roux and Martin, who have specially studied this procedure,112have shown that a horse may be easily immunized by injecting into the animal the toxin diluted with a third of its volume of Gram's iodine solution, and in successively increasing doses. The initial dose is 0.25 Cc.; then, after two days, 0.5 Cc. of the same toxin is injected, and in like manner the dose is increased up to the eighteenth day, when the pure toxin is injected, at first in small doses, which are gradually increased so that at the end of two or three months injections of 80 Cc. of the pure toxin may be given without danger; the animal is then completely immunized.
The serum of an animal rendered immune in this manner contains a diphtheria antitoxin which possesses high power. A guinea-pig which has received an injection of 0.01 Cc. of the antitoxin is perfectly capable of withstanding a lethal dose of 0.5 Cc. of the toxin. The antidiphtheria serum thus obtained, and in almost limitless quantities, from an immunized animal, is capable of saturating the therapeutic diphtheritic toxin, and has to-day taken rank in therapeutics as the most efficacious remedy in diphtheria. Injected in varying doses, it confers a temporary but immediate immunity.
Nevertheless antidiphtheria serum must not be considered as an antidote; and in pathological diphtheria, the more serum is required the later it is used.113In certain cases, if employed too late, it may prove ineffective.
The preventive action of the serum is remarkable. In 10 000 inoculated cases Behring and Ehrlich have had but 10 cases of diphtheria, and these were, moreover, of a benign character. The duration of the immunizing action appears to be from three weeks to two months.
This diphtheria antitoxin was first prepared by Guérin and Macé114by adding to the antidiphtheria serum a large volume of alcohol, washing the precipitate, and drying it in a vacuum. It is soluble in water, and loses its activity when heated to 65° C. Wassermann115has proposed to extract it from the milk of immunized animals, by first coagulating the milk by rennet in the presence of sodium chloride, filtering, and removing the fat from the clear liquid by means of chloroform. After decanting, the clear solution obtained is precipitated by adding to it 30 to 33 per cent. of ammonium sulphate. The precipitate is dried in a vacuum on a polished porcelain slab after havingfirst been strongly expressed. It is then dissolved in water.116
Tetanus Toxin.—The fact that the tetanus bacillus never penetrates to the interior of the organism enabled us long ago to foretell that it secretes a very powerful toxin capable of dialyzing and diffusing through the economy. Kuno Faber was the first to fully recognize the fact that the culture bouillon of this bacillus, fully sterilized by filtration through porcelain, possesses an exceedingly high toxicity, and exerts a toxic effect on 50 000 000 times its own weight of living organism. Brieger had previously, however, extracted three ptomaines from the cultures of the bacillus—tetanin,tetanotoxin, andspasmotoxin.117In order to obtain a highly active liquid, the same culture medium is inoculated several times in succession, but filtering each time before the new inoculation; the microbes greatly increase in number after each fresh inoculation, and the toxic substance developed by them accumulates.118
Experiment has shown that the culture bouillon thus obtained contains two kinds of toxic substances119—highly toxic alkaloidal bases (ptomaines, tetanin, tetanotoxin, etc.), and a true toxin, possessing diastatic properties, and of almost incredible toxic power.
This toxin had already been isolated by Kitasato. It is a toxalbumin, and is very sensitive to the action of heat. A temperature of 65° C., maintained for 30 minutes, renders it quite inactive; and it becomes oxidized and is destroyed by the action of the air in the presence of light.
Brieger and Boer,120by precipitating with zinc chloride the filtered culture bouillon, obtained a pure, amorphous tetanus toxin, which they also considered as a toxalbumin, and which possesses exceedingly toxic properties.
If a precipitate be caused to form in these toxic solutions, as, for instance, a precipitate of calcium phosphate, this carries down with it all the toxin present in the liquid. 0.0005 Gm. of this precipitate is surely fatal to a guinea-pig.
Dozon and Cournemont have observed that even in doses of 300 to 400 Gm. of the filtered culture liquid, this toxin is not immediately toxic to a horse, but kills the animal only after a period of incubation of at least twenty-four hours. The blood of such an animal, however, is immediatelyand directly fatal to animals into which it is injected.121
Experiment has shown that animals that have been cured of tetanus possess no immunity whatever against tetanus; nevertheless Behring and Kitasato122first, and Wassermann and Kitasato later on, succeeded in preparing atetanus antitoxin. To obtain this, the immunization of the animal, horse or cow, is effected by injecting increasing quantities of the toxin, more or less attenuated by mixing it with Gramm's iodine solution; the immunization is easily and rapidly accomplished by the process devised by Roux and Vaillard.123
The immunized animals yield a serum which, mixed with tetanus cultures, renders these innocuous, and which enjoys an antitoxic power that borders on the marvelous.124A quintillionth of a cubic centimeter of the serum per gramme weight of a live mouse suffices to protect the animal from an otherwise fatal quantity of tetanus toxin.125
This serum is nevertheless powerless to preserve man in cases of acute tetanus; it confers an immediate, but only transitory, immunity.
As to its mode of action, it appears to cause a permanent condition of excitation or of nutritive reaction of the cells, which makes these resistant to the poison. As in the case of the other toxins, the quantity of antitoxin necessary to protect an organism is so much greater the later the treatment is applied.
Mallein (Toxin of Glanders).—Among the soluble products secreted in the culture media by the glanders bacilli, there are found true toxins to which are ascribed certain symptoms of glanders infection. These toxins have been isolated and designated by the namemallein. First prepared by Helman and Kalmino, mallein was later on specially studied by Roux and Nocard, and, in consequence of the researches of the last-mentioned scientist, it has acquired great importance.126It is obtained by sterilizing at 110° C. cultures of the glanders bacillus made with mutton bouillon with the addition of salt, glycerin, and peptones. To isolate the toxin the culture bouillon is first sterilized by heating for half an hour in an autoclave at 100° C. It is then filtered, concentrated to one-tenth its volume on a water-bath, and filtered through a Chardin filter. The mallein is thus obtained in the form of a brown syrupy liquid containing half its weight of glycerin.
This solution keeps well when kept from air, light, and heat. In practice it is employed in 10-percent. solution in phenolated water (5:1000). The mallein may be precipitated from the crude solution by the addition of alcohol, as recommended by Foth. Foth's mallein occurs as a white, light powder, very easily soluble in water.
Mallein enjoys a very important rôle in veterinary therapeutics, a rôle analogous to that of tuberculin, permitting the diagnosis of incipient glanders.127
Experience has shown that in animals already attacked by glanders, even if ever so slightly, the thermic reaction never fails when 0.25 Cc. of the mallein solution is injected. In healthy animals, however, the injection of mallein, even in much larger quantities, causes no apparent effect. In animals attacked by glanders the reaction attains its maximum in twelve hours, and several days are required for the temperature to return to normal.128
According to Nocard, mallein possesses no immunizing properties whatever.129
Typhoid Toxin.—This is obtained, like the other microbial toxins, from a culture, prepared with more or less difficulty, from Eberth's typhoidbacillus. This toxin, injected into guinea-pigs, develops in them typhoid fever.
In the solution there occurs a ptomaine, which has been isolated by Brieger, and which gives rise to almost all the phenomena of typhoid fever; this ptomaine is calledtyphotoxin.130
The same author, in collaboration with Fraenkel,131later on isolated a toxalbumin from the culture bouillon of the typhoid bacillus. Sanarelli132obtained an active toxin by macerating for several days at 60° C. a month-old culture of the typhoid bacillus made with a 2-per cent. glycerin-bouillon. Chantemesse has also published a process which yields a highly virulent toxin.133
Chantemesse and Widal134have shown that on injecting into an organism increasing quantities of the sterilized cultures of Eberth's Bacillus, it is possible to fully immunize an animal against the bacillus itself, and even also against the Bacillus coli communis. The operation, however, is tedious and painful. The serum of immunized animals possesses preventive and curative properties respecting the effects of typhoid bacilli.
A dose of the filtered culture, which is fatal to a guinea-pig, becomes innocuous when mixed with 0.5 Cc. of the serum of a vaccinated guinea-pig; 6 Cc. of the serum injected six hours after an injection of the virulent culture, hence when this is in full action, suffice to save the animal.135So far as the human being is concerned, the results obtained have not been sufficiently satisfactory.
The culture bouillon of the Bacillus coli communis, which is closely allied to Eberth's bacillus, also contains soluble toxic substances which have been named coli-bacillus toxin. This substance, which is produced only in small quantity by the microbe, is fatal only in very large doses.
Cholera Toxin.—Very little is known regarding the toxic products of the spirillium choleræ; nevertheless, the fact that typical cholera exhibits every symptom of the action of a toxic agent demonstrates quite clearly the elaboration of some toxic substance within the cultures of this microbe.
Villiers136found in it a liquid ptomaine; Klebs137found another and crystallizable ptomaine; while Pitai discovered in it a toxin unalterable by heat, and which he considered as a toxopeptone. According to Gamaleia138there is present a true toxin,alterable by heat, and the reactions of which entitle it to be considered as a nucleo-albumin; he has also found in it a toxic nuclein.
These toxic substances are found, according to Gamaleia, Pfeiffer, and Sanarelli,139confined during the life of the microbe within its cellular envelope, and does not diffuse through this. Metchnikoff and Roux are of the contrary opinion,140however, and they have prepared a toxin almost insensitive to a temperature of 100° C., and precipitable from its solutions by ammonium sulphate or strong alcohol; the toxin is a toxalbumin. This toxin is quite toxic; one-third of a cubic centimeter suffices to kill 100 Gm. of guinea-pig in 18 hours; with larger doses, death is almost immediate.
By immunizing guinea-pigs, rabbits, and horses with this cholera toxin, Metchnikoff and Roux obtained a serum which is distinctly antitoxic for rabbits. Nothing absolutely certain has been found as to its action on man.141
We will not dwell longer here on the toxins of microbial origin. It appears evident, however, from what has been stated above, that the greatmajority, if not all, of the virulent microbes manifest their virulence by means of toxic secretions. Almost every one of these toxins has been the subject of study. They would otherwise not have interested us here, where our main object was but to dwell upon the general properties.