Guinea pigs were inoculated with suspensions of Leptospira icterohæmorrhagiæ obtained from pure cultures of several different strains, in order to determine whether or not an active immunity against a subsequent infection with virulent organisms would develop in the vaccinated animals. The experiments were so arranged as to make possible a determination of the existence of immunity against homologous strains as well as against the strains not employed as vaccine, and a brief quantitive estimation of the degree and duration of the immunity in relation to the quantities of the vaccines inoculated. Following the general rule of prophylactic inoculations with various pathogenic organisms, the inoculations were repeated subcutaneously on three consecutive occasions at intervals of 5 days. With respect to the amounts of vaccine, the experiments were divided into three groups for each vaccine, one group receiving three doses of 0.5 cc., the second three of 0.05 cc., and the third three of 0.005 cc. Four different strains were employed as vaccines, American Strain 1, American Strain 2, and one each of the Japanese and the European strains.
The determination of the development, degree, and duration of the immunity was made by inoculating intraperitoneally several minimum lethal doses of each of the five following strains: American Strains 1, 2, and 3, the Japanese, and the European strains. The virulence of the different strains varied considerably, the strongest being the Japanese strain, which killed the guinea pig in a dose of 0.00001 cc., and the weakest American Strain 3, the minimum lethal dose of which was as large as 0.01 cc.
The vaccinated guinea pigs were tested for immunity at the end of 2, 4, and 8 weeks after the last inoculation.
The results obtained show that three successive inoculations of 0.5 cc. of the emulsions of killed cultures of Leptospira icterohæmorrhagiæ into guinea pigs rendered them completely resistant to a subsequent infection with the virulent cultures of both homologous and heterologous strains. With 0.05 cc. the protection was not so general, the animals succumbing to an experimental infection with some heterologous strains while resisting the homologous and other heterologous strains. The animals which were vaccinated with 0.005 cc. survived the infection experiments with the homologous strains in the case of American Strain 1 and the Japanese strain, but they were not protected against any other strains. The vaccines of other strains were unable to immunize the guinea pigs so highly even against their homologous strains, when the amount of each inoculation was only 0.005 cc., but 0.05 cc. conferred complete protection against the same strains. It may be concluded, therefore, that when a sufficient quantity of killed cultures of Leptospira icterohæmorrhagiæ is given, the guinea pigs will become immune to all strains of the same organism, but that smaller quantities may protect them against homologous but not against heterologous strains. A close analysis reveals the existence of group or type affinities among different strains which can be brought ' out by immunizing the animals with smaller quantities of killed cultures. In the present series of experiments American Strains 1 and 3 form one group, American Strain 2 and the European strain another, and the Japanese strain a third, which is also closely allied to the first group.
In order to insure universal immunity it is wise to employ as many group or type cultures as possible in the preparation of vaccines, a polyvalent vaccine being recommended. It is not improbable that the strain recently encountered in Lorient, France, is an unusually deviated type of Leptospira icterohæmorrhagiæ, and that if successfully cultivated and used as vaccine in sufficient amount it might protect the animals against other strains of the same organism.
The active immunity induced in the vaccinated guinea pigs was found to persist for at least 8 weeks after the last inoculation. It will no doubt last for a much longer period.