Experiments were carried out for the study of culture spirochetes in their relation to various immunity reactions in vitro. Several strains of Treponema pallidum and one each of Treponema calligyrum, Spirochata refringens, Treponema microdentium, and Treponema mucosum were used. Tests were made of immune substances responsible for agglutination, complement fixation, spirocheticidosis, and opsonization. In cases of agglutination and complement fixation, cross titrations were made.

1. In the sera derived from rabbits immunized with various spirochetes agglutinins were demonstrated in varying quantities for the homologous antigens. The amounts of agglutinins developed were considerably higher in the pallidum immune sera than in the other groups. There was no parallelism between the amounts of antigens injected and the amounts of agglutinins developed.

2. Cross titrations among different pallidum strains revealed that the agglutiantion is not necessarily strongest when homologous antigens and immune sera are brought together.

3. On the other hand, the reactions between the immune sera and antigens belonging to different species were sufficiently specific to justify the grouping.

4. Certain degrees of group reactions were observed between the pallidum immune sera and the calligyrum, and occasionally very faintly also between the pallidum and the refringes antigens and vice versa. There was a much more pronounced group reaction between the calligyrum and refringes. The immune serum and antigen of the microdentium showed a slight affinity for the mucosum but none for the pallidum, calligyrum, or refringes, while the mucosum immune serum caused a slight agglutination with many members of the other groups. Hence, it appears that the pallidum is more or less related to the calligyrum, while the affinity between the calligyrum and refringes, and possibly also between the calligyrum and mucosum in a much smaller degree, seems close. The microdentium showed the least relation to any other spirochetes.

5. Titration of agglutinins in the sera obtained 3 months after the cessation of immunization revealed that the agglutinin contents were already greatly reduced, having fallen roughly to 0.01 of the original strenght. The rates of disappearance were irregular in different animals and bore no direct relation to the initial titers. Titration made of the immune sera which had been preserved aseptically in a refrigerator (6°C.) during the same period (3 months) indicated that the original strength of these sera was reduced to about one-tenth. The agglutinins for spirochetes disappear from the rabbit's body much more rapidly than they are reduced in the separated sera by deterioration on standing at 6°C.

6. Titration of the immune sera for complement fixation power showed with a few exceptions, in which there was only slight complement binding, that the titers were high enough to indicate the presence of this principle. The anti-pallidum sera possessed higher average titers than the other immune sera tested with correspondingly homologous antigens. The least active were the anti-refrigens sera.

7. Cross titration of anti-pallidum immune sera for complement fixation showed that a given serum with a high titer for its own strain of antigen was also strong with most of the other strains of the pallidum. Instances occurred also in which the titers with heterologous pallidum antigens fell far below those of the homologous. Group reactions between the different spirochetes) such as the pallidum and the calligyrum, the calligyrum and the refringens, and the microdentium and the mucosum, were also indicated. The mucosum and the pallidum showed a slight degree of group reaction. No anti-pallidum serum fixed complement with the microdentium.

8. The immune sera were tested for their spirochetiddal properties in vitro against the correspondingly specific and heterologous varieties with and without the addition of complement. Many of the anti-pallidum sera killed their own strains. Normal rabbit serum exhibited only a slight degree of inhibition. Without complement, the immune sera caused a considerable reduction in the number or density of colonies, but not a complete suppression of growth. Complement alone had no injurious effect upon the pallidum strains. The antisera for the calligyrum, refringens, and mucosum showed feeble spirocheticidal action, while the antisera for microdentium was stronger. A syphilitic rabbit serum tested against a strain of culture pallidum gave a feeble inhibitory effect.

9. Under the influence of immune sera and complement, the spirochetes undergo within a few hours complete disintegration or granular degeneration. Without complement, they are more powerfully agglutinated, but no disintegration occurs, even after 20 hours, and complement alone has no effect.

10. In the presence of homologous immune serum and complement, the culture pallidum may be ingested by the leukocytes, but phagocytosis is slight, possibly on account of the filamentous nature of the organisms. The spirochetes in such a mixture disintegrate within a few hours, disintegration being especially rapid when the immune leukocytes are used. In the absence of immune serum, phagocytosis is not noticeable, while without complement but in the presence of immune serum and leukocytes, some phagocytosis, without subsequent lysis, occurs.

A virulent strain of pallidum, obtained from syphilitic orchitis in a rabbit, exposed to agglutination, lysis, and phagocytosis by an immune serum prepared by means of culture pallidum strains, showed only slight agglutination and phagocytosis but rapid immobilization without disintegration in the presence of complement.

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