ARTIFICIAL IMMUNIZATION 229 



and also from two asses, Calmette next made a most important stride in the 

 antivenin treatment of human beings in snake poisoning. Whatever errors 

 he may have made as to the interpretation of the mechanism of passive 

 immunity and the specificity of antivenin, medical science owes to Calmette 1 

 much of the development and achievement of this particular phase of venom 

 immunity. 



In 1895-1896 he had immunized a number of horses against venom by 

 the usual processes. He started with small, gradually increasing doses of 

 the solution of cobra venom mixed with small, gradually decreasing quantities 

 of 1 : 60 solution of calcium chloride, injected subcutaneously, with an inter- 

 val of 4 or 5 days between each injection. In general, after 2 months' im- 

 munization the horses became able to bear a dose of pure venom which could 

 kill 100 kilograms of rabbit. After reaching this stage the horses showed less 

 and less local reaction to the injection and their serum attained a considerable 

 degree of antivenomous power. Not less than six months was necessary 

 before the serum could be of sufficient potency for therapeutic purposes. 

 Therefore a longer time is required for venom immunization than for 

 diphtheria toxin. Even after a large number of injections the local oedema 

 followed each inoculation and persisted for many days. The horses became 

 uneasy and lost their appetite, their respiration was accelerated and inspira- 

 tory, they perspired abundantly, but had no fever. These symptoms usually 

 disappeared in 2 or 3 days. If the injection was too frequently repeated the 

 horse had nephritis, haematuria, and eventually succumbed. The choice of 

 venom was of some importance and Calmette employed the venom of Cobra 

 because it is the most active of all venoms and has less local effect than the 

 other venoms, such as the venom of Pseudechis of Australia. 



Calmette mentions that the phylogenetic principles of various venoms of 

 Viperidse are by no means identical, because sometimes intense haemolysis 

 occurs, while at others simply a white oedema follows the injection of this 

 group of venoms. These local poisons are completely destroyed by 75° C. 

 or a small quantity of calcium chloride. When the immunization of the 

 animal advanced pretty high in regard to the cobra venom, Calmette injected 

 various kinds of venoms of other species, thus avoiding the violent sloughing, 

 haemorrhage, or oedema which would have followed if these venoms had been 

 given without the preliminary immunization with cobra venom. When the 

 animal became able to stand the injection of a dose of venom fatal to 500 

 kilograms of rabbit, he judged the immunization to be complete enough. 



The standardization of the antivenomous power of Calmette was made on 

 rabbit, whereas an antivenin capable of neutralizing or preventing the effect 

 of 1 minimal lethal dose per 1.000 gm. of rabbit by the dose of 0.1 c.c. of the 

 antivenin was expressed in numerical value of 10.000. This means that 1 c.c. 

 of such serum can neutralize the minimal lethal dose of venom for 10.000 gm. 

 of rabbit. Calmette believed that a serum possessing 10.000 antivenomous 



• Calmette. Le venin des serpents. Physioloj*ie de l'envenimation, traitement des morsures veni- 

 meuses par le serum des animaux vaccines. Soc. d'eciit. scientif., Paris, 1896. 



