264 THE DIPHTHERIA BACILLUS 



2nd day of experiment. Injection of 0*5 c.c. of an iodized toxin. No local 

 nor general reaction. 



4th, 6th, and 8th days of experiment. Injection of 0*5 c.c. of an iodized toxin. 

 No local nor general reaction. 



13th and 14th days of experiment. Injection of 1 c.c. of an iodized toxin. No 

 reaction. 



17th day of experiment. Injection of 0'25 c.c. of a pure toxin. Slight oedema. 

 No rise of temperature. 



22nd day of experiment. Injection of 1 c.c. of a pure toxin. Slight oedema. 

 No rise of temperature. 



23rd day of experiment. Injection of 2 c.c. of a pure toxin. Slight oedema. 

 No rise of temperature. 



25th day of experiment. Injection of 3 c.c. of a pure toxin. Slight oedema. 

 No rise of temperature. 



28th day of experiment. Injection of 5 c.c. of a pure toxin. Slight oedema. 

 No rise of temperature. 



30th, 32nd, and 36th days of experiment. Injection of 5 c.c. of a pure toxin. 

 Slight oedema. No rise of temperature. 



39th and 41st days of experiment. Injection of 10 c.c. of a pure toxin. Slight 

 oedema. No rise of temperature. 



43rd, 46th, 48th and 50th days of experiment. Injection of 30 c.c. of a pure 

 toxin. Fairly well marked oedema which vanished in 24 hours. 



53rd day of experiment. Injection of 60 c.c. of a pure toxin. Fairly well 

 marked oedema which vanished in 24 hours. 



57th, 63rd. 65th, and 67th days of experiment. Injection of 60 c.c. of a pure 

 toxin. Fairly well marked oedema which vanished in 24 hours. 



72nd day of experiment. Injection of 90 c.c. of a pure toxin. Fairly well 

 marked oedema which vanished in 24 hours. 



80th day of experiment. Injection of 250 c.c. of a pure toxin. Fairly well 

 marked oedema which vanished in 24 hours. 



This horse therefore had received in 2 months and 20 days 800 c.c. of toxin, with- 

 out showing any symptoms other than a transient local oedema, some loss of appetite 

 and a rise of temperature of about 1 C. on the evenings following the larger injec- 

 tions. The animal was bled on the 87th day and was inoculated into the jugular 

 vein with 200 c.c. of toxin without showing any reaction. 



Vaccinated horses withstand equally well enormous doses (many hundred cubic 

 centimetres) of living cultures. 



As has been pointed out some horses are more susceptible to diphtheria toxin 

 than others, and in the more susceptible individuals an extensive, firm oedema 

 lasting many days may follow inoculation, and in some cases, the horse may sweat 

 and show a marked rise of temperature. 



Occasionally a highly immunized horse will die of paralysis 1-3 weeks after the 

 last inoculation of toxin. 



With the very powerful toxins at present in use immunization should be 

 carried out still more carefully. With a toxin containing 200 units of toxin 

 per c.c. horses should be inoculated three times a week for 6 weeks, with a 

 mixture of toxin and Gram's solution (commencing with a mixture consisting 

 of 2 parts Gram's solution and 1 part toxin), then with toxin alone in pro- 

 gressively increasing doses : the initial dose being 0'5 c.c. and the final 

 inoculation 200 c.c. 



When small doses are inoculated at frequent intervals the antitoxic content 

 of the serum is greater than when large doses are given at longer intervals 

 (Roux). 



To maintain horses in a state of immunization it is necessary to inoculate 

 a dose of toxin from time to time : this may be done in different ways. 



1. After bleeding a horse 300-500 c.c. of culture may be inoculated at 

 intervals of 20 or 25 days into the jugular vein. 



2. At the Pasteur Institute Martin inoculates beneath the skin of the 

 shoulder, 13 days after bleeding the animal, 300 c.c. of toxin ; and 4 days 

 later on the opposite side also sub-cutaneously a further 500 c.c. The 

 horse may be bled again a week after the last inoculation. 



