582 PROCEEDINGS OF SECTION G2. 



experimental cases by the bacilli. There seems to be a consider- 

 able resistance to the invasion of the blood-stream during life, as 

 in the allied disease, blackleg ; but immediately prior to death, 

 and especially soon after death, the bacilli multiply readily and 

 very rapidly within the blood as do other anaerobic putrefactive 

 bacteria ; hence the general state of putrefaction with emphy- 

 sema in which the cadaver is usually found. 



Diminution of Virulence in Cultures and Immunization 

 BY such Cultures. 



This occurs very definitely in all media, but much more so in 

 glucose media than in ordinary broth or serum broth. If sub-cultures 

 be made from day to day on ordinary serum broth, the virulence may 

 be retained through a number of successive generations. Should, how- 

 ever, inoculations from tube to tube be only made at intervals of several 

 days the virulence is gradually lost. For example, a sheep (lamb 

 3) inoculated with 0.3 cc. of a sixth sub-culture three days old, 

 which had been transferred at irregular periods for two months 

 from tube to tube of serum broth, showed no marked sign of illness 

 for 36 hours, and death occurred only fully three days after inocu- 

 lation, the post-mortem picture being typical. Another sheep 

 (sheep 4) inoculated with 0.5 cc. of the ninth sub-culture five days 

 old showed slight swelling with lameness and rise of temperature 

 the following day, after which recovery was rapid. Another sheep 

 (lamb 5) inoculated with 0.5 cc. of a tenth sub-culture, four days 

 old (transferred at intervals during three months) remained normal 

 but for a transient swelling. Similar experiments carried out on 

 guineapigs gave like results, and further inoculation with virulent 

 material indicated that a certain degree of resistance was acquired 

 as a result of inoculation with these weakened cultures. 



More definite and more rapid weakening of virulence was found 

 to be obtained by cultivation in gulcose broth, both with and 

 without serum. 



For example, 0.15 cc. of such cultures, when a day old, proved 

 fatal for guineapigs in less than 20 hours, yet seven days later the 

 same doses of the same cultures proved innocuous for similar 

 guineapigs. 



These results suggested further experiments on sheep, the 

 following being the results : — 



Tasmanian Bacillus. — Sheep 19 was inoculated with 3 cc. 4th 

 sub-culture, seven days old, in glucose serum broth. Result : 

 Remained normal, but for passing lameness and slight rise of tem- 

 perature. Fourteen days later this animal proved immune to 0.25 

 cc. first sub-culture on ordinary serum broth, while a control sheep 

 inoculated with a similar dose of the same culture succumbed. 



Another sheep (26) was inoculated with 5 cc. second glucose 

 serum broth sub-culture seven days old. Result : Remained normal 

 but for slight swelling and rise of temperature. A guineapig 

 inoculated with 1 cc. of the same culture developed a shght swelUng, 

 from which it rapidly recovered. In these cases, however, immunity 



