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 named diphtheritic 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 Yersin[110] were 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 this toxin[111] a 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,[112] have 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.