530 THE ANTHRAX BACILLUS 



Before death symptoms of paraplegia appear, respiration is laboured and the animal 

 lies on its side and may have convulsive attacks. 



Death may take place at any time between the 2nd and 15th or 20th day after 

 inoculation, the duration of life depending upon the amount of toxin inoculated. 



Guinea-pigs and mice are susceptible to the toxin. 



Contrary to Hankin's experience animals immune to anthrax are almost 

 unaffected by the toxin : this is also true with rabbits immunized with 

 attenuated cultures. 



The toxin is weakened but not completely destroyed by heating it to 

 110 C. The addition of alkaline hypochlorites, chloride of gold, or Gram's 

 solution destroys its toxic properties, as does also prolonged exposure to 

 sunlight in presence of air. 



Cultures of anthrax in other liquid media (ox serum or broth made with beef, 

 veal or horse) contain but little toxin. Marmier was able to extract an active 

 toxin from recent cultures on agar: the growth from 48 -hour old cultures on agar 

 was scraped and macerated in alcohol containing a few drops of ether at 20 C. ; 

 after autolyzing for 24 hours the emulsion was filtered and the filtrate precipitated 

 with absolute alcohol : the precipitate on the filter was washed with absolute alcohol 

 then with ether and finally dried in vacuo over sulphuric acid. The powdery product 

 was as toxic as the toxin prepared from cultures in glycerin-peptone-water. This 

 indicates that primarily the toxins are intra- cellular. 



Vaccination with Toxin. 



I. Toussaint conferred immunity on sheep by inoculating them with 

 denbrinated anthrax blood heated to 55 C. for 10 minutes. 



Better results are obtained if the anthrax blood be heated to 60 C. on 

 three or four different occasions before injecting the sheep. A slight degree 

 of immunity is obtained in this way which disappears after a lapse of time 

 varying from a month to 3 years (Roux and Chamberland). 



II. Hankin stated that with a toxin which he had prepared by growing 

 the bacillus in broth made with Liebig's extract and containing fibrin, animals 

 could be easily immunized against anthrax. After certain objections had 

 been raised by Peterman, Hankin reinvestigated the subject and obtained 

 less satisfactory results. If the inoculation of toxin in amounts equal to 

 i~Wooo f kdy weight has any immunizing action at all on mice it must 

 be of a transitory nature and in any case only a small proportion of animals 

 treated in this way resist the test inoculation. 



HI. Marmier succeeded in immunizing laboratory animals with a toxin 

 prepared by growing the bacillus in glycerin-peptone-water. Rabbits after 

 being repeatedly inoculated with small doses acquired a certain degree of 

 immunity which however did not last longer than 5 or 6 weeks after the 

 last inoculation. It is nevertheless possible to immunize rabbits against 

 doses of toxin which if given to untreated animals would cause their death. 



The method is as follows : The rabbit is first of all inoculated with a very small 

 dose of this toxin, for example 3 mg., and when it has recovered, that is to say in 

 about 6 days, a larger dose (6 mg.) is administered and when the reaction has sub- 

 sided a third dose of 15 mg. is given. About 12 days after the third inoculation the 

 animal can, in the majority of cases, survive the inoculation of a virulent culture 

 of anthrax. By gradually increasing the amount inoculated to 20 and 30 mg. 

 immunity can almost certainly be ensured. Care must be taken to see that the 

 animal has completely recovered from the effects of the immunizing inoculations 

 before giving the test inoculation. 



4. Serum therapy. 



I. Behring has shown that the serum of white rats exhibits bactericidal 

 properties against the anthrax bacillus. When a small quantity of a culture 



