528 THE ANTHRAX BACILLUS 



Pasteur's method of vaccination. The more severe the vaccinating infection 

 the more highly immunized will the animal be. 



On the other hand there is the danger that if a powerful vaccine be inocu- 

 lated in the first instance the animal may die and the experiment have to 

 be recommenced. 



In practice a compromise is effected by using two vaccines. The first 

 inoculation is made with a very weak vaccine which will kill mice but has 

 no ill-effect on rabbits and sheep (premier vaccin). The second inoculation 

 is given 12 days later : it is somewhat more virulent than the first vaccine 

 and will kill mice and guinea-pigs and, twice out of six or eight times, 

 rabbits (second vaccin). Immunity is established 12 days after the second 

 inoculation. 



The vaccine prepared by the Institut Pasteur is supplied to veterinary 

 surgeons at a trifling cost in tubes containing 100 doses. The first and 

 second vaccines are inoculated successively, the dose for a cow being 0'25 c.c. 

 and for a sheep 0'125 c.c. 



As a rule, the inoculations are given as follows : The first vaccine is inocu- 

 lated into the internal surface of the right thigh and the second into the 

 internal surface of the left thigh, an interval of 12 days elapsing between 

 the two operations. The vaccine must be used as soon as it is procured and 

 the inoculations must be done with a sterile syringe. It is important not to 

 use a contaminated vaccine, because by contamination it has lost its properties. 



Immunization of small animals. The immunization of small animals is 

 difficult but necessary for laboratory investigations. Rabbits and guinea- 

 pigs are so highly susceptible that death often occurs during the process. 

 For such animals three vaccines of different virulence ought to be available, 

 the first very attenuated, the second the premier vaccin and the third the 

 second vaccin described above. The inoculations should be carried out very 

 cautiously. 



Marchoux succeeded in immunizing laboratory animals by using only the 

 " sheep vaccines." These vaccines were grown at 37 C. in a peptonized 

 veal broth and used when 24 hours old. The first vaccine given under the 

 skin of a rabbit consisted of 0'5 c.c. (maximum non-fatal dose) of the more 

 attenuated of the two sheep vaccines. It caused a rise of temperature, 

 diarrhoea and loss of weight. The second vaccine given 12 days later con- 

 sisted of a double dose of the same vaccine. The third inoculation given 

 after another interval of 12 days consisted of 0*25 c.c. of the more virulent of 

 the sheep vaccines (second vaccin) and 12 days later again a fourth inoculation 

 of 0'5 c.c. of the latter vaccine was inoculated. A week after the last inocula- 

 tion the animal was tested by the inoculation of a few drops of anthrax blood 

 under the skin. If the reaction was not too violent the immunizing process 

 was continued and completed by the frequent inoculation of anthrax blood 

 or of virulent cultures 24 hours old. Marchoux succeeded in immunizing 

 some of his rabbits to such a degree that they were able to resist the daily 

 inoculation of 1 c.c. of fully virulent bacilli while other rabbits could resist 

 the inoculation every fifth day of gradually increasing doses up to 20 c.c. 



Immunization by avirulent cultures. De Christmas showed that white rats can 

 be immunized with totally avirulent cultures. He injected into the peritoneal 

 cavity of white rats 1 c.c. of a watenr emulsion of a young avirulent culture on 

 three different occasions at intervals^ of a month. The material for sowing the 

 cultures was taken from a non-sporing strain which had been grown for a number 

 of years at the Institut Pasteur and which was totally devoid of virulence for white 

 rats. All the animals vaccinated in this manner were inoculated a month after 

 the last immunizing inoculation with a large dose of a virulent anthrax bacillus 

 which was certainly fatal to control animals, and were found to be immune. 



