In a previous paper (Metzger and Podwiinska, 1967) evidence was presented that the development of agglutinability which occurs in ageing suspensions of T. pallidum (Hardy andNell, 1955, 1957; Metzger and Podwinska, 1965) (2) Each batch of treponeme suspensions under (i), (ii), and (iii), prepared anew for every immunization week, was tested for virulence by injecting 01 ml. of the suspension containing 29 million organisms at each of four sites on the shaven back of a rabbit, and 0 5 ml., 299

The results reported in a previous paper (Metzger, Michalska, Podwiriska, and Smog6r, 1969) have clearly shown that the immunogenic properties of Treponema pallidum are related to their protein component. A high degree of immunity to challenge inoculation by virulent treponemes was obtained in rabbits that had been injected with killed treponemes with the protein antigen preserved, as judged by their high agglutinability in a serum that contained antibody to this component only. Two vaccine preparations proved very efficient when given intravenously to rabbits:(1) Treponemes maintained without any additions at 4°C. for from 7 to 10 days before administration.(2) Treponemes stored for the same time but with peniciUin added (4 .tg./ml.).The main purpose of the present study was to check the efficiency of these preparations when administered intramuscularly and to investigate the effect of treponeme dose on the development of resistance. Material and MethodsThe virulent Nichols strain of T. pallidum was used throughout.Adult Danish albino rabbits weighing about 3 kg., with non-reactive VDRL and TPI tests, were used for the study; they were housed in individual cages out of doors. VaccinesThree vaccines prepared from killed T. pallida were given as immunizing agents:(1) Treponemes suspended in phosphate-buffered saline of pH 7.4 and stored at 4°C. for from 7 to 10 days. *Received for puiblication April 21, 1969 tThis study was supported by grants from the,World Health Orztion. 308(2) Treponemes suspended in phosphate-buffered saline of pH 7.4 with penicillint added (1 Vg./ml.); stored at 37'C. for 24 hours and at 4°C. for 6 to 9 days.(3) Treponemes suspended in phosphate-buffered saline of pH 7-4 with penicillin added (4 jLg./ml.); stored at 370C. for 24 hours. Non-infectiousness of the vaccines was ascertained by the following experiments:(1) Each new batch of treponeme suspensions was tested for virulence by intracutaneous injection of 0.1 ml. containing 29 million organisms into each of four sites on the shaven back of a rabbit, and by injection of 05 ml. containing 145 million organisms into each testis of the same rabbit. None of the rabbits developed lesions during a 4-month observation period, and lymph node transfers from these rabbits to other rabbits were negative. All the animals remained VDRL and TPI negative.(2) 4 or 5 weeks after the completion of the immunization procedure, the left popliteal lymph node of each animal was removed and emulsified in 1 ml. 10 per cent. normal rabbit serum-saline; one drop of the emulsion was inspected by darkfield microscopy for the presence of treponemes, and the rest was injected into one testis of a normal rabbit; the second testis was utilized for lymph node transfer from another rabbit. Microscopic examination of the testes performed one month later did not show the presence of treponemes in any of the testicles. ImmunizationOnly female rabbits were used. They were injected intramuscularly with various vaccines over a period of 7 weeks. Each rabbit received 1 ml....