330 THE TUBERCLE BACILLUS 



endotoxin. This extract, or acido-toxin, is made by macerating tubercle bacilli in a 

 10 per cent, aqueous solution of ortho-phosphoric acid for 2 hours at 60 C., neutraliz- 

 ing the product, filtering and diluting nineteen times with water (1 in 20 solution of 

 endotoxin). 



3. Vaccination. 

 A. Laboratory animals. 



(i) Grancher and Martin. Rabbits are inoculated with avian tubercle 

 bacilli which have been grown on artificial media for prolonged periods. By 

 using successively less and less attenuated cultures they have succeeded in a 

 few cases in producing a certain degree of immunity in the animals. 



(ii) Hericourt and Richet sterilize cultures of avian bacilli by heating 

 them several times to 80 C. and then inoculate them into rabbits in doses of 

 10-20 c.c. This method has enabled them to confer immunity on a few 

 animals. 



(iii) Courmont and Dor filter glycerin-broth cultures and inoculate the 

 filtrate into rabbits at the same time that or before they inoculate a tuber- 

 culous virus. With bacilli of avian origin they succeeded in producing 

 immunity twice in four experiments but they failed with the bacillus of 

 human origin. 



(iv) In the foregoing experiments, attempts to vaccinate guinea-pigs had 

 failed. E. Levy immunizes guinea-pigs by inoculating them with a tubercle 

 bacillus [which he states to have been] attenuated by being kept in glycerin 

 (p. 322). 



Two guinea-pigs are inoculated, one into the peritoneal cavity, the other sub- 

 cutarftously, with a slightly opalescent emulsion of tubercle bacilli which has been 

 kept in 80 per cent, glycerin for 6 days in the incubator at 37 C. When they have 

 recovered from the first inoculation they are again inoculated on successive occasions 

 with bacilli which have been kept in glycerin for 4, 3 and 2 days. When they have 

 completely recovered they, as well as two control animals, are inoculated with a 

 tubercle bacillus of standard virulence. All the animals develop an abscess at the 

 site of inoculation but at the end of about 4 weeks the lesion in the case of the 

 vaccinated guinea-pigs has healed while in the controls the disease has spread to 

 the glands. Towards the end of the third month the controls are suffering from a 

 generalized tuberculosis (liver, spleen and lungs) while the most minute search 

 fails to reveal any trace of tuberculosis in the vaccinated animals killed at the same 

 time. 



B. Cattle. 



(i) Von Behring succeeded in vaccinating young bovine animals (healthy 

 calves under one year old) by inoculating them intra-venously with living 

 attenuated bacilli of human origin, non-virulent for cattle (bovo-vaccin). 



The bacillus used by von Behring in his experiments was a human tubercle bacillus 

 which had been in artificial culture for 8 years and had lost much of its original 

 virulence (vide p. 322, attenuation of tubercle bacilli). A five- week-old culture of 

 this bacillus oh glycerin -serum could be inoculated with impunity into the veins of 

 a calf in doses of 0'005 gram. A first injection of 0*001 gram provoked no reaction 

 and was followed by several other inoculations with increasing doses at intervals of 

 several weeks. Animals treated in this way were finally able to resist doses of 

 bacilli of bovine type which were fatal to the control animals. 



Behring has now modified his original procedure. He first inoculates, intra- 

 venously, 0'004 gram of a glycerin-agar culture of a bacillus of human origin com- 

 pletely dried in vacua at the ordinary temperature (the bacilli are ground up in a 

 mortar and emulsified in 4 c.c. of a 1 per cent, saline solution). A month later 

 the animal is similarly inoculated with 0'01-0'02 gram of the same culture. 



Animals treated in this way and exposed to contagion or inoculated with a virulent 

 bacillus of bovine origin never develop any tuberculous lesion. And, further, 

 when tested with tuberculin a year after immunization they always fail to react. 



