THE PROBLEM OF VIRULENCE 17 



beyond which it could no longer be enhanced. After only three 

 passages through monkeys, however, the virulence of this "virus 

 fixe" for rabbits was reduced almost to extinction. His experience 

 with swine plague was similar. Swine plague bacilli successively 

 passed through rabbits and pigeons gained enormously in virulence 

 for these animals respectively, but lost in virulence for hogs. 



There are numerous methods by which the virulence of micro- 

 organisms can be attenuated by laboratory manipulations, and since 

 many of them are of great importance in the active immunization of 

 animals we will reserve their detailed discussion until we come to 

 consider the methods of immunization themselves. Suffice it to say 

 in this place that most methods of attenuation consist in subjecting 

 the bacteria, in artificial culture, to deleterious influences, either of 

 unfavorably high temperature, exposure to light or harmful chemi- 

 cal agents, or allowing them to remain in prolonged contact with the 

 products of their own metabolism by infrequent transplantation. As 

 a rule the attenuation which inevitably follows any form of arti- 

 ficial cultivation in the case of bacteria like streptococci or pneumo- 

 cocci can be delayed by preserving them in media containing sera 

 or tissues. In the case of the pneumococcus, for instance, one of 

 the best methods of conserving virulence in storage is to keep them 

 either in a soft rabbit-serum-agar mixture, as practiced by Wads- 

 worth, or, better still, to store them within the spleen of a mouse 

 dead of pneumococcus infection, as recommended by Neufeld. The 

 mouse is autopsied and the spleen kept in the dark and cold in a des- 

 iccator, under sterile precautions. This, again, as well as the en- 

 hancement of virulence on passage through the same species of ani- 

 mal or the reduction of virulence for one species by passage through 

 another shows that such fluctuations are dependent upon a very 

 delicate biological adaptation. 



It is interesting, moreover, to look upon this process of adapta- 

 tion as a sort of immunization of the bacteria against the defensive 

 powers of the host, a conception early suggested by Welch. For just 

 as the animal body may become more resistant to the offensive 

 weapons of the invaders, so it is reasonable to suppose that the bac- 

 terial body may gradually develop increased resistance to the de- 

 fensive mechanism of the host. And this, if it occurs, would of 

 course lead to an increase of its invasive power or virulence. The 

 increase of virulence by passage through animals would alone lead 

 us to suspect that such acquired resistance to destructive agents on 

 the part of the bacteria might be responsible for the enhancement, 

 but additional evidence pointing in this direction has been brought 

 by experiments in which it was shown that bacteria cultivated in the 

 serum of immune animals not only gained in resistance to destruction 

 by the serum constituents, but at the same time were rendered more 

 highly pathogenic. Experiments of this kind were carried out by 



