VACCINATION 587 



twice their weight of water for 24 hours, a few pieces of thymol being added as an 

 antiseptic. The fluid is then filtered several times through paper and finally through 

 a Chamberland bougie. 



(iii) From the blood of rabbits killed by his virulent virus (vide ante) Issaeff 

 obtained a toxin capable of killing rabbits on intra-venous inoculation when 

 administered in a dose equivalent to one one-hundredth of the weight of the 

 animal. The toxicity of the product is considerably diminished by heating it 

 to 70 C., and is destroyed at 100 C. His method is as follows : 



1. Collect under aseptic precautions the heart- blood of three or four rabbits 

 which have recently succumbed to the inoculation of a virulent pneumococcus 

 (80-100 grams of blood) and mix the various samples in a sterile vessel. 



2. Add an equal volume of sterile water containing 1 per cent, of glycerin and 

 5 or 6 drops of a saturated solution of bicarbonate of sodium per 100 c.c. Mix. 



3. Filter the mixture through a Chamberland bougie. 



The toxicity of the product is much diminished by heating to 70 C. and destroyed 

 at 100 C. 



By filtering pleural and peritoneal exudates of rabbits which had died after 

 the inoculation of a virus of increased virulence Issaeff was also able to obtain 

 a product sufficiently toxic to kill rabbits. 



4. Vaccination. 



(i) With toxins. It is possible to immunize an animal against the pneumo- 

 coccus by inoculating it with filtered cultures or with toxins prepared by 

 the methods of Emmerich, Mosny or IssaefT ; the immunity however is of 

 short duration, and to render the animal more permanently immune it must 

 be inoculated afterwards with living cultures. 



A. Heat the serum of rabbits which have died of a pneumococcal infection to 

 58 C. and inoculate it in doses of 1020 c.c. into the ear vein of a fresh rabbit. After 

 four or five inoculations at intervals of a few days the animal is able to resist the 

 inoculation of virulent cultures (Foa). 



B. A similar result is obtained by inoculating in the same way toxins prepared 

 by the methods of Emmerich or Mosny. 



C. Issaeff immunizes rabbits by inoculating into the veins at intervals doses 

 of 10-50 c.c. of sterilized cultures (broth or serum). Each inoculation is followed 

 by a fairly sharp reaction so that the next inoculation must be withheld until the 

 animal appears to have completely recovered from the previous experiment. 



The same observer has also immunized rabbits by inoculating them with toxins 

 extracted from the blood as described above. A single injection of 10 c.c. of toxin 

 into the blood or peritoneal cavity is sufficient to render rabbits highly immune 

 against the pneumoccocus. 



The immunized animals are tested by inoculating them sub-cutaneously with the 

 blood of a rabbit just dead of a pneumococcal infection ; on the first occasion 2-4 

 drops and on the second 0*5 c.c. of the blood are administered. To keep up the 

 immunity the animal should be re-inoculated once a month with a dose of not more 

 than 0'5 c.c. sub-cutaneously. Before giving the test inoculation it is necessary 

 to wait until the animal has completely recovered from the effects of the immuniza- 

 tion and until the weight has begun to increase. 



D. Neufeld and Handel immunized horses by inoculating them intra-venously 

 with organisms killed by heat. Highly virulent cultures in broth were heated to 

 60 C. and centrifuged. The organisms alone were inoculated : the animals tolerated 

 the inoculations very well even when they were repeated in very large doses at fre- 

 quent intervals. The resulting serum was rich in thermostable substances which 

 assisted the phagocytosis of virulent pneumococci ; a dose of 2 c.c. intra-peritoneally 

 immunized mice against the inoculation of 0*1 c.c. of a very virulent culture. 



Rabbits vaccinated with toxin are absolutely immune to infection with 

 living cultures, but not to toxins, to which they react even more violently 

 than do normal rabbits. 



