522 PROTOZOA 



and more convincing fact has been demonstrated by Huddleston 

 and others, namely, that when active vaccine is mixed in certain pro- 

 portions with serum from an animal which had just recovered from a 

 successful vaccination, and the mixture is inoculated into a susceptible 

 animal, there is no reaction. 



Etiology of Variola and Cowpox. It has been repeatedly shown that 

 no bacteria similar to any of the known forms have a causal relation 

 to these diseases. In our own laboratory we are able, by the inoculation 

 of rabbits' skins, to produce extremely active vaccine virus in large 

 quantities, absolutely free from micro-organisms as grow under the con- 

 ditions of our present methods of bacterial cultivation. Such pure active 

 vaccine, when emulsified in equal parts of glycerin and water and fil- 

 tered through two or three thicknesses of the finest filter paper, gives a 

 slightly opalescent filtrate, which in the hanging drop under high magni- 

 fication shows many very tiny granules with an occasional larger one, 

 and in smears shows no formed elements giving characteristic stains. 

 This filtrate, from which no growth can be obtained on artificial culture 

 media, when rubbed over a freshly shaved rabbit's skin, after the 

 method of Calmette and Gue*rin, gives an abundant typical reaction. 



These facts show that some, at least, of the infective forms cannot as 

 yet be made to grow outside of the body, that such forms are very tiny, 

 and that they do not stain characteristically with our usual methods of 

 staining. We have shown also that the infective agent cannot pass an 

 ordinary Berkefeld filter under forty pounds' pressure, which practi- 

 cally rules out ultramicroscopic forms. 



Since Guarniere in 1892 claimed that certain inclusions present in 

 the epithelial cells of the lesions of smallpox in a rabbit's cornea (Fig. 

 160) were parasites, much attention has been given to the study of these 

 bodies, commonly known as "vaccine bodies," yet opinions still differ as 

 to their nature. The most recent studies of importance of these bodies 

 have been made, on the one hand, by Councilman and his associates, who 

 believe them to be protozoa, and, on the other, by Ewing, who believes 

 that all of the forms so far described are degeneration products, some 

 specific, others not. 



Councilman believes that there are two cycles of development of the 

 "parasite," one intracellular and the other intranuclear, arid that the 

 intranuclear infection occurs only in smallpox. The intracellular cycle 

 is simple, showing only "multiplicative reproduction," while the intra- 

 nuclear cycle is more complicated, probably sexual in character. 

 Calkins, working with Councilman, has described an elaborate cycle 

 of development in which we believe are included many forms due to 

 degeneration of the host cells alone. 



In our own work on sections, which has extended irregularly over a 

 period of several years, we have gotten results which are somewhat con- 

 fusing, principally so because of the non-uniformity of the appearances 

 of these bodies, both by different methods and by the same methods at 

 different times. There is no doubt that whatever the nature of the bodies 

 they are easily affected by methods used for fixing, hardening, and staining 



