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’* 
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, hematuria, 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 
Viperide are by no means identical, because sometimes intense hemolysis 
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, 
hemorrhage, 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 
ee a eae ee eee SS OO 
1Calmette. Le venin des serpents. Physiologie de Venvenimation, traitement des morsures veni- 
meuses par le sérum des animaux vaccinés. Soc. d’édit. scientif., Paris, 1896. 
