rpp( 



IMMUNIZATION OF ANIMALS 263 



received from 10-20 c.c. of these cultures at various times. Immunity was 

 acquired at the end of 14 days. 



(c) Behring in immunizing guinea-pigs and rabbits used the pleural fluid 

 obtained from guinea-pigs which had succumbed to the inoculation of virulent 

 cultures. Immunity was acquired in about a fortnight after the inoculation 

 of the vaccine, but the results were very inconstant. 



(d) Behring immunized guinea-pigs and sheep by inoculating them with 

 cultures 3 weeks old and to which 1 part of iodine terchloride had been added 

 to 500 parts of culture. A few c.c. of this mixture were inoculated into an 

 animal and then 10 days or so later a second inoculation was given of a 

 culture to which a smaller quantity of iodine terchloride had been added. 

 Immunity was acquired in about a fortnight. This method fails in the case 

 of the rabbit. 



(e) Brieger, Wassermann and Kitasato conferred immunity on guinea- 

 pigs by inoculating them on several occasions with 2 c.c. of a culture on 

 thymus broth warmed to 70 C. for 15 minutes. But this method is not so 

 effective as the iodine terchloride method of Behring. 



(ii) Koux, Nocard and Martin succeeded in immunizing various animals 

 (rabbits, sheep, goats, cows, horses) by inoculating them first with a virulent 

 toxin mixed with Gram's iodine solution, then with gradually increasing 

 doses of pure toxin. 



A rabbit, for example, was inoculated sub-cutaneously in the first instance with 

 0*5 c.c. of the following mixture which was prepared immediately before use. 



Toxin (Roux and Martin's method), 2 vols. 



Gram's iodine solution, ..... 1 vol. 



The injection was repeated every few days for some weeks, then the proportion 

 of iodine was gradually diminished and last of all pure toxin was inoculated. The 

 animals were weighed at frequent intervals and if they showed any loss of weight 

 the inoculations were stopped for the time being, otherwise they died of cachexia. 



Goats and cows may be immunized in a similar manner, but these animals being 

 very susceptible to diphtheria toxin, very small doses of iodized toxin must be 

 used for the initial inoculations, and pure toxin should only be given when the 

 blood shows some content of antitoxin. It should be borne in mind that pregnant 

 animals are more susceptible to diphtheria toxin than non-pregnant animals. 



Horses stand toxin well and especial interest attaches to the immunization 

 of these animals because they are the source whence antitoxin for thera- 

 peutic purposes is derived. 



The horses selected should be young (6-9 years old) well fed and free from 

 disease. After having been tested with mallein to exclude a possible infection 

 with glanders (vide Glanders) [and with tuberculin to exclude tuberculosis] 

 the horse is inoculated in the first instance with a small quantity of a virulent 

 toxin to which Grani's iodine has been added : at subsequent inoculations 

 the doses are gradually increased, and after the eighth inoculation pure toxin 

 is used : different animals vary enormously in susceptibility, and care should 

 always be taken that the dose used in the initial experiment shall be so small 

 that no violent reaction results, as this might imperil the steady progress 

 of the immunizing process. The injections should be made sub-cutaneously 

 into the neck or behind the shoulders. 



The following table exhibits an actual record of the immunization of an horse by 

 Roux and Nocard. 



Horse, 7 years old and weighing about 400 kg. 



The iodized toxin contained one-tenth its volume of Gram's solution. The toxin used 

 killed guinea-pigs weighing 500 grams in 48 hours in doses of O'l c.c. The injections 

 were made beneath the skin of the neck or behind the shoulder. 



1st day of experiment. Injection of 0*25 c.c. of an iodized toxin. No local 

 nor general reaction. 



