Fig. 94.—Aseptically Bleeding a Horse Vaccinated against Venom in order to obtain Antivenomous Serum at the Pasteur Institute at Lille.
When a horse is well vaccinated and tolerates without a reaction 2 grammes of dry cobra-venom in a single subcutaneous injection, it may be bled on three consecutive occasions in the space of ten days, and in this way 20 litres of blood may be drawn from it ([fig. 94]).
The bleeding is arranged in the following manner: Twelve days after the last injection of venom the horse is bled for the first time to the extent of 8 litres; five days later it is bled for the second time to the extent of 6 litres; five days later still the third bleeding takes place, when 6 litres are again withdrawn.
The animal is then allowed to rest for three months and supplied with strengthening food, and during this period 2 grammes of venom are again injected on two occasions at the end of a month, followed, a month and a half later, by the injection of 2 more grammes. The antitoxic power of the serum is thus maintained approximately at the same standard.
The serum drawn off at each bleeding must be severely tested, which is done by gauging its antitoxic power in vitro, when mixed with venom, and also its preventive effect.
An antivenomous serum may be considered to be utilisable when a mixture of 1 c.c. of serum with 0·001 gramme of cobra-venom produces no intoxicating effect in the rabbit, and when a preventive subcutaneous injection of 2 c.c. of serum into a rabbit of about 2 kilogrammes enables it to resist, two hours later, subcutaneous inoculation with 1 milligramme of venom.
The preventive power may be very quickly tested by injecting a rabbit, in the marginal vein of the right ear for example, with 2 c.c. of serum, and injecting, five minutes afterwards, in the marginal vein of the left ear, 8 milligramme of venom. This dose of 1 milligramme generally kills the control rabbits in less than thirty minutes when introduced into the veins, and in from two to three hours when injected beneath the skin.
This rapid proof by intravenous injection is extremely striking and demonstrative; it can be effected in public during a class or lecture in less than an hour, and enables an immediate estimate to be formed of the value of an antivenomous serum. When it is intended to adopt this method, it is essential to make use of a recent solution of venom, for solutions from a week to a fortnight old, although sterile, have already lost a large portion of their toxicity, and, if these be employed, the dose of venom calculated to kill the control animals in thirty minutes, for example, takes an hour or more to do so.
I always prepare my test solutions of venom in the following manner:—
Ten milligrammes of dry cobra-venom are weighed in a delicate balance. The venom is dissolved in 10 c.c. of 0·8 per cent. physiological salt solution, which takes a few minutes. When the venom is thoroughly dissolved it is transferred to a test-tube, which is immersed for three-quarters of an hour in a water-bath heated to + 72° C. In this way the non-toxic albumins are coagulated without modifying the neurotoxic substance. The solution is poured on to a filter of sterilised paper, and the clear liquid which is collected is immediately put up in glass phials, which are hermetically sealed, or in small sterilised bottles. Its toxicity is tested upon control animals, and it may be kept for five or six days if protected from light, or for several weeks in a refrigerator at about 0° C.