Identity of Bacillus parabotuUnus. 161 



0.0005 c.c. per kilogramme body weight led to death in 63 hours, 

 and other products have shown almost as high a titre. Even so it is 

 not believed that the limits of toxicity have been reached, and further 

 enquiry is being made into the factors associated with maximal toxin 

 production. 



The evidence presented in our earlier paper that B. parahotulinus 

 produced a true toxin was as follow: — 



1. The administration of germ-free filtrates lead to symptoms 



identical with those induced by whole cultures. (These filtrates 

 were obtained after passage of culture through Chamberland F. 

 or Doulton candles, and were tested culturally for sterility.) 



2. Such symptoms ensued only after a well marked period of in- 

 cubation, varying according to dosage and route of administra- 

 tion. This period of incubation was commonly of from 15 

 hours to several days; even after intravenous inoculation of 

 large doses into guinea-pigs no symptoms were manifested for 

 at least six hours. 



3. The administration of small doses of such filtrates sufficed 



to set up the characteristic symptoms and death. 



4. The toxicity of a filtrate was diminshed on heating to 60° C. 



for 15 minutes and entirely destroying by heating to 80° C. 

 for a like period. 



5. The fact that filtrates possessed a toxic property analogous to 

 that of botulinus toxin, i.e., were toxic when administered by 

 the mouth. 



It was admitted that as no antitoxin had up till then been pre- 

 pared against these toxic filtrates, the claim that a true specific exo- 

 toxin was present in these cultures could not be wholly substantiated. 



Production of Antitoxin. 



Repeated attempts have been made to immunise small animals 

 against the toxic filtrates of B. parahotuUnus. In addition to these 

 two horses have also been employed. The first of these horses had 

 to be discontinued for another cause, and the second horse has received 

 .as yet only comparatively few injections; a test of its serum reveals 

 the presence of antibodies, but its serum is as yet of too low a potency 

 for critical experiment.* 



At first gradual doses of unalter^ed toxin, starting with a small 

 fraction of a lethal dose, were used, but without success, and the 

 animals (guinea pigs and rabbits) invariably succumbed sooner or 

 later as the doses were increased. 



For the next attempt toxin heated to 60° C. for one hour was em- 

 ployed for the earlier injections, and by this means, two guinea pigs 

 out of six have been successfully immunised. The remainder of these 

 guinea pigs died during the immunisation process; one of those 

 immunised at one time exhibited marked symptoms of paralysis, from 

 which, however, it gradually recovered; the other likewise showed 



* This horse has now been successfully immunised. January, 1923. H.K.S. 



