Four hours after his death, the mucus from the palate of the child was collected. It was diluted with a little water, and two rabbits were inoculated under the skin of the abdomen. The rabbits perished in less than thirty-six hours. The saliva of these dead rabbits also transmitted the disease to fresh rabbits. Did it not seem as if one had got hold of an inoculation of hydrophobia? Such was in fact the conclusion of Dr. Maurice Raynaud, who, having been informed, at the same time as Pasteur, of the illness of the child, had made, on his own account, some experiments on rabbits. His rabbits were dead. Already a year previously M. Maurice Raynaud had announced the transmission, by the saliva, of rabies from man to rabbits. 'We are, then, in the presence of a new fact of this kind,' he said, 'and we really believe, until a proof to the contrary is given us, that these latter rabbits died of hydrophobia.'
With his habitual prudence, and trusting more to the results of experiment than to medical observation alone, Pasteur was not in a hurry to form such positive conclusions. He began by doing what Dr. Maurice Raynaud had neglected to do. He examined with the microscope the tissues and the blood of the rabbits inoculated in the laboratory; he discovered, both in those that were dead, and in those which were on the point of death, the presence of a special microbe, easily cultivable in a pure state and of which the successive cultures caused the death of other rabbits. Invariably, the same microbe appeared in the blood. As one or two days sufficed to cause death, hydrophobia could not have had time to make its appearance. Pasteur, moreover, found this same microbe in the saliva of children who had died of common maladies, and even in the normal saliva of healthy adults. It was a new microbe, causing a disease unknown up to that time. To Pasteur it seemed, in the case of the experiments made with the mucus from the child's palate, to be simply an accompaniment of the rabic virus.
This microbe of the saliva is very easily cultivated in sterile infusions—that of veal, for example—and successive cultures can be made in the usual way. The virulence continues. Could the virulence be attenuated, asked Pasteur, by the action of the oxygen of the air? This would, by a new example, go to establish the generalisation of the method of attenuation by oxygen. The attempt succeeded. When care is taken, as with the attenuation of the fowl cholera contagium, not to allow more than some hours' interval to elapse between one cultivation and the succeeding one, the virulence of the successive cultivations of the microbe of the saliva is preserved in some sort indefinitely. In other words, if it be arranged that the cultures succeed each other every twelve hours, the rabbits inoculated from the last cultures die as quickly as those inoculated from the first. Thuillier had had the patience to make, in this manner, eighty cultures in contact with air, and eighty cultures in a vacuum; the microbe of the saliva being both aerobic and anaerobic. The eightieth culture killed as quickly as the first. But by allowing the successive cultures to remain for some time in contact with the air, before passing from one culture to the following one, the virulence of the cultures becomes enfeebled. Thus, then, as in fowl cholera, attenuated cultures of the microbe can be obtained. Unlike what happens with cholera, however, the cultures of the microbe of the saliva, exposed to the contact of air, perish very quickly. Two or three days of keeping suffice for the parent cultivation to lose all virulence. The seed, taken in any quantity from it, does not fertilise a new cultivation. But, before perishing, this culture passes through very different degrees of progressively weakened virulence, and it is easy with these cultivations to render rabbits ill without causing their death. Once cured, they resist all inoculation which would be mortal for others. The oxygen of the air is manifestly the transformer of this virulent virus into vaccine virus; for, if the virulent blood or cultivations remain inclosed in their tubes, sealed from all entrance of the air, they retain not only for some hours, but for months, their life and their original virulence.
But though these results were as new as they were unexpected, and though one cause of confusion in the study of this terrible problem was removed, yet these first researches were not marked by any progress in the etiology of hydrophobia. The question remained wholly unsolved.
Impatient with the length of time required for the incubation of the disease, and with the obligation of waiting whole months for the result of an experiment, when the subject demanded such numerous ones, Pasteur began to seek some means of producing hydrophobia with certainty and of making it appear more rapidly. Notwithstanding the assertion of a professor of the veterinary school at Lyons that the saliva of the rabid dog alone contained the virus of the disease, and that he had failed in every attempt to inoculate, whether with the substance of the brain or with the spinal marrow of rabid dogs; Pasteur, with due care as to purity, introduced under the skin of some rabbits and some dogs, divers parts of the brain of a dog which had died in a rabid state. Hydrophobia declared itself in both dogs and rabbits, with a duration of incubation about equal to that which followed the ordinary bite of a dog. Although it was necessary still to submit to this long uncertainty with regard to the incubation, one great result was obtained: hydrophobia could be inoculated with other matter than saliva. Not only is the saliva always impure, containing a saliva microbe, which is endowed with a special virulence of its own, but it presents other inconveniences. It is necessary, in these researches, to have a supply of material constantly at hand. Now, the saliva loses its rabic virulence in twenty-four hours. The existence of the rabic virulence in the brain substance placed, on the contrary, at the disposal of the experimenter, an abundance of the virus, in a state of great purity and capable of long preservation.
The idea then occurred to Pasteur and his assistants, to inoculate the virulent rabic matter in its pure state under the dura mater on the surface of the brain of a dog. Why not carry the virus, said Pasteur, directly to the place of its activity and development? After having trepanned the skull of a chloroformed dog, a little bit of the medulla of an animal which had died of hydrophobia was deposited on the surface of the brain. As soon as the influence of the chloroform was dissipated, the dog recovered its healthy appearance. It ate its food that same evening. But after some days the symptoms of hydrophobia appeared. The animal became dejected and restless; it tossed its litter about, refused all nourishment. A doleful, sharp howling was the first indication of the rabic voice, which is but one long cry of suffering and appeal, mingled with barkings from hallucinations. The stomach became depraved; the dog swallowed hay and straw. It soon grew furious, agitated with violent convulsions; finally, after a last fit, it died. During all this time there was great rejoicing in the laboratory. They were at last in possession of a method for singularly shortening the period of incubation, and for communicating the disease with certainty. The experiments were multiplied; all the dogs which were trepanned, and which received on the surface of the brain a little of the medulla of the rabid animal, succumbed to the disease, with very rare exceptions, within a period of twenty days. Did not the method pursued demonstrate, among other things, that hydrophobia is a disease of the brain; that the seat of the rabic virus, far from being exclusively in the saliva, belongs, above all, to the cerebral matter?