We have seen how the facts have been established with regard to the microbe of fowl cholera. Immunity against a virulent disease may be obtained by the influence of a benign malady which is induced by the same microbe, only weakened in virulence. What a future there would be for medicine if this method could be applied to the prevention of all virulent diseases! As splenic fever was at that time being studied in the laboratory of the École Normale, it was upon this fever that the research was first attempted. But the success of this research, said Pasteur, can only be hoped for if the disease is non-recurrent. It is only in this case that inoculation with the weakened microbe can protect from the deadly splenic fever. Unfortunately, human medicine is dumb as regards this question of non-recurrence. The man who is smitten with malignant pustule rarely recovers. If there are any cases of recovery—and there are some authentic ones—he who has so narrowly escaped death could not confidently count upon his chance of protection from the disease in future. In order to acquire such a sense of security he would have to expose himself to experiments of direct inoculation, which he would hardly care to do. Animals alone offer the possibility of solving this problem. Yet it is not to all species of animals that we can have recourse. Every sheep inoculated with splenic fever infection is a sheep lost; but the ox and the cow have more power of resistance. Among them there are frequent cases of cure. An incident occurred which enabled Pasteur to push very far this experimental study.

In 1879 the Minister of Agriculture appointed him to give judgment upon the value of a proposed mode of cure for cows smitten with splenic fever, which had been devised by M. Louvrier, a veterinary surgeon of the Jura. Choosing M. Chamberland as his assistant to watch the application of M. Louvrier's remedy, Pasteur instituted a series of comparative experiments. Some cows were inoculated, two and sometimes four at a time, with the virulent splenic fever virus. Half of these cows were treated by M. Louvrier's method; the other half were left without treatment. A certain number of the cows under M. Louvrier's care resisted the disease, but an equal number of those not under treatment recovered also. The inefficacy of the remedy was demonstrated as well as the cause of the inventor's illusions. But one precious result remained from the trial of this remedy. Pasteur and Chamberland had thus at their disposition several cows which had recovered from splenic fever, and which had experienced in their attack all the worst symptoms. At the places of inoculation enormous swellings were formed, which extended to the limbs, or under the abdomen, and which contained several quarts of watery fluid. The fever had been intense, and at one time death had appeared imminent. When these cows recovered they were reinoculated with great quantities of virulent virus. Not the least trace of disease showed itself, even in cases where the inoculation was performed after an interval of more than a year.

The question was solved; splenic fever, like most of the virulent diseases which it has been possible to study, was non-recurrent. The immunity obtained has a long duration. With that valiant ardour which always urges him on, Pasteur next proposed to examine the vaccine of splenic fever. In view of these new investigations, which would require long and careful labours, and which necessitated a certain amount of medical knowledge, Pasteur associated with himself, in addition to M. Chamberland, a young savant, now a doctor of medicine, M. Roux.

Following the rigorous course of his deductions, Pasteur naturally turned to the oxygen of the air in his attempts to modify the virulence of the splenic microbe. But a difficulty presented itself at the outset. Between this microbe and the microbe of fowl cholera there exists an essential difference. The microbe of fowl cholera, as is the case with a great number of microscopic organisms, reproduces itself only by fission. The parasite of splenic fever, on the contrary, has another mode of generation; it forms spores, nothing analogous to which is found in the microbe of cholera.

In the blood of animals, as in the cultures at the beginning, the splenic fever microbe appears at first in transparent filaments, more or less divided into segments. Up to that point, the resemblance between the microbe of splenic fever and the microbe of cholera is complete. But this blood, or the cultures exposed to the free contact of the air, instead of continuing this first mode of generation, frequently exhibit, even in the course of twenty-four hours, spores distributed more or less regularly along the length of the filaments. All around these corpuscles the matter of the filaments is absorbed, in the manner formerly illustrated by Pasteur in the diagrams of his work on the diseases of silkworms, when treating of the bacilli of putrefaction. Little by little, all cohesion between the spores disappears, and the whole collection soon forms nothing more than a dust of germs. But—and here lies the great difficulty which experimenters encountered in applying to splenic fever the method of gradual attenuation which was practised with the microbe of cholera—these germs of splenic fever may be exposed for years to the air without losing their virulence, always ready to reproduce themselves without any appreciable change, and to manifest their effects in the bodies of animals. How can it be hoped to discover a vaccine of splenic fever by the method used with the contagium of fowl cholera, since the splenic fever virulence, at the end of twenty-four hours, is concentrated in a spore? Before the oxygen of the air has had time to attenuate the contagium, the virulence of the parasite would be encased in these spores. Yet this objection did not appear insuperable to Pasteur. Since (said he to himself), under its filamentous form, the microbe of splenic fever is quite analogous to the microbe of fowl cholera, may not the problem of exposing the splenic microbe to the air be reduced to the following one: to determine the conditions which would prevent the production of spores? The difficulty would thus be surmounted; for, once we have got rid of the spores, the splenic filaments might be maintained in contact with air for any length of time, and we might then no doubt fall back upon the conditions which had produced the attenuation of the cholera microbe.

Pasteur and his two assistants gave themselves up to this research. Days passed and experiments were multiplied. Pasteur became more and more engrossed: he had, what his daughter called, 'the face of an approaching discovery.'

'Ah! what a grand thing it would be,' he was heard from time to time to murmur to himself with a suppressed voice, 'if one could arrive at that—if the fact that the attenuation of the microbe of fowl cholera proved not to be an isolated one!' But if anyone ventured to ask him a timid question as to the phase his experiments were going through, he would reply, 'No, I can tell you nothing. I dare not express aloud what I hope.'

At last one day he came up from his laboratory with a triumphant face. His joy was such that tears stood in his eyes. I have never seen a more radiant expression of the highest and most generous emotions than emanated from his countenance.

'I should never console myself,' he said while embracing us, 'if a discovery such as my assistants and I have just made were not a French discovery.'

And with the clearness which is the charm of this powerful mind, he related to us the most recent discoveries of his laboratory.

In neutralised chicken infusion the splenic microbe can no longer be cultivated at a temperature of 44 or 45 degrees. Its cultivation, on the contrary, is easy at 42 or 43 degrees; and in these conditions the microbe produces no spores. At this latter temperature, therefore, and in contact with pure air, we can maintain a culture of filamentous parasites of splenic fever, deprived of all germs. In some weeks the crop dies—that is to say, when this culture is sown in fresh broth the sterility of the broth remains complete. But during the preceding days life exists in the cultivating liquid. If after two, four, six, or eight days of exposure to the air and to heat, the contagium is tried upon animals, its virulence is found to be continually changing with the time of its exposure to the air, and, consequently, it represents a series of attenuated contagia. From the moment when the formation of the spores of the splenic fever bacillus is prevented, all becomes substantially the same as in the case of the microbe of fowl cholera. Moreover, as in the cholera microbe, each of these states of attenuated virulence can be reproduced by cultivation. Finally, splenic fever not being recurrent, each of these splenic fever microbes constitutes a vaccine for the more virulent microbe.

In order to apportion the virulence of the vaccine to the species it is desired to vaccinate, it must be tried on a certain number of individuals of the same species. If some vaccinated animals are inoculated with the virulent virus, and none of them perish, the vaccine is good. Among individuals of the same species, however, the difference of receptivity is in general great enough to make it prudent, and even necessary, to have recourse to two vaccines, one weak and the other stronger, with an interval of from 12 to 15 days between the two inoculations.

It was on February 28, 1881, that Pasteur communicated to the Academy of Sciences, in his own name, and in those of his two fellow workers, the exposition of this great discovery. Loud applause burst forth with patriotic joy and pride. And yet so marvellous were these results that some colleagues could not help saying, 'There is a little romance in all this.' All this reminds one, in fact, of what the alchemist of Lesage did to the demons which annoyed him. He shut them up in little bottles, well corked, and so kept them imprisoned and inoffensive. Pasteur shut up in glass bulbs a whole world of microbes, with all sorts of varieties which he cultivated at will. Virulences attenuated or terrible, diseases benign or deadly, he could offer all. Hardly had the journals published the compte-rendu of this communication when the President of the Society of Agriculture in Melun, M. le Baron de la Rochette, came, in the name of the Society, to invite Pasteur to make a public experiment of splenic fever vaccination.

Pasteur accepted. On April 28 a sort of convention was entered into between him and the Society. The Society agreed to place at the disposal of Pasteur and his two young assistants, Chamberland and Roux, sixty sheep. Ten of these sheep were not to receive any treatment; twenty-five were to be subjected to two vaccinal inoculations at intervals of from twelve to fifteen days, by two vaccines of unequal strength. Some days later these twenty-five sheep, as well as the twenty-five remaining ones, were to be inoculated with the virus of virulent splenic fever. A similar experiment was to be made upon ten cows. Six were to be vaccinated, four not vaccinated; and the ten cows were afterwards, on the same day as the fifty sheep, to receive inoculation from a very virulent virus.

Pasteur affirmed that the twenty-five sheep which had not been vaccinated would perish, while the twenty-five vaccinated ones would resist the very virulent virus; that the six vaccinated cows would not take the disease, while the four which had not been vaccinated, even if they did not die, would at least be extremely ill.

As soon as the agricultural and scientific press had published this programme, and recorded Pasteur's prophecies, several of his colleagues at the Academy of Sciences, startled by such boldness in reference to a subject which had hitherto been enveloped in such profound obscurity, and fearing to see the illustrious company somewhat compromised by these affirmations in relation to problems of physiology and pathology, addressed some observations to M. Pasteur on what they called 'a scientific imprudence.'

'Take care,' they said to him, 'you are committing yourself without possibility of retreat. Your experiments in the laboratory hardly authorise you to attempt experiments like those at Melun.'

'No doubt,' Pasteur answered, 'we have never had in our experimental studies so many animals at our disposition to inoculate; but I have full confidence. What has been already done in my laboratory is to me a guarantee of what can be done.'

And M. Bouley, confident also in the assurances of his illustrious friend, and arranging to meet him, to witness these audacious experiments, said to his anxious colleagues, 'Fear nothing; he will come back triumphant.'

The experiments began on May 5, 1881, at four kilometers' distance from Melun, in a farm of the commune of Pouilly-le-Fort, belonging to a veterinary doctor, M. Rossignol, secretary-general of the Society of Melun. At the desire of the Society of Agriculture, a goat had been substituted for one of the twenty-five sheep of the first lot. On the 5th of May they inoculated, by means of the little syringe of Pravaz—that which is used in all hypodermic injections—twenty-four sheep, the goat, and six cows with five drops of an attenuated splenic virus. Twelve days after, on May 17, they reinoculated these thirty-one animals with an attenuated virus, which was, however, stronger than the preceding one.

On May 31 very virulent inoculation was effected. Veterinary doctors, inquisitive people, and agriculturists formed a crowd round this little flock. The thirty-one vaccinated subjects awaiting the terrible trial stood side by side with the twenty-five sheep and the four cows, which awaited also their first turn of virulent inoculation. Upon the proposal of a veterinary doctor, who disguised his scepticism under the expressed desire to render the trials more comparative, they inoculated alternately a vaccinated and a non-vaccinated animal. A meeting was then arranged by Pasteur and all other persons present for Thursday, June 2, thus allowing an interval of forty-eight hours after the virulent inoculation.

More than two hundred persons met that day at Melun. The Prefect of Seine-et-Marne, M. Patinot, senators, general counsellors, journalists, a great number of doctors, of veterinary surgeons, and farmers; those who believed, and those who doubted, came, impatient for the result. On their arrival at the farm of Pouilly-le-Fort, they could not repress a shout of admiration. Out of the twenty-five sheep which had not been vaccinated, twenty-one were dead; the goat was also dead; two other sheep were dying, and the last, already smitten, was certain to die that very evening. The non-vaccinated cows had all voluminous swellings at the point of inoculation, behind the shoulder. The fever was intense, and they had no longer strength to eat. The vaccinated sheep were in full health and gaiety. The vaccinated cows showed no tumour; they had not even suffered an elevation of temperature, and they continued to eat quietly.

There was a burst of enthusiasm at these truly marvellous results. The veterinary surgeons especially, who had received with entire incredulity the anticipations recorded in the programme of the experiments, who in their conversations and in their journals had declared very loudly that it was difficult to believe in the possibility of preparing a vaccine capable of triumphing over such deadly diseases as fowl cholera and splenic fever, could not recover from their surprise. They examined the dead, they felt the living.

'Well,' said M. Bouley to one of them, 'are you convinced? There remains nothing for you to do but to bow before the master,' he added, pointing to Pasteur, 'and to exclaim—

"I see, I know, I believe, I am undeceived."'

Having suddenly become fervent apostles of the new doctrine, the veterinary surgeons went about proclaiming everywhere what they had seen. One of those who had been the most sceptical carried his proselytising zeal to such a point that he wished to inoculate himself. He did so with the two first vaccines, without other accident than a slight fever. It required all the efforts of his family to prevent him from inoculating himself with the most virulent virus.

An extraordinary movement was everywhere produced in favour of vaccination. A great number of agricultural societies wished to repeat the celebrated experiment of Pouilly-le-Fort. The breeders of cattle overwhelmed Pasteur with applications for vaccine. Pasteur was obliged to start a small manufactory for the preparation of these vaccines in the Rue Vauquelin, a few paces from his laboratory. At the end of the year 1881, he had already vaccinated 33,946 animals. This number was composed of 32,550 sheep, 1,254 oxen, 142 horses. In 1882, the number of animals vaccinated amounted to 399,102, which included 47,000 oxen and 2,000 horses. In 1883, 100,000 animals were added to the total of 1882.

From the commencement of the practical application of this new system, the results were topical. Among flocks where half had been vaccinated and the other half not vaccinated—all the animals continuing to live together—the mortality from splenic fever in 1881 was ten times less in the vaccinated sheep than in the non-vaccinated, being 1 in 740 as against 1 in 78; and in cows and oxen fourteen times less, being 1 in 1,254 against 1 in 88. In 1882 also, the mortality was ten times greater among the non-vaccinated than among the vaccinated animals.

In 1883 it was proved that the duration of the immunity generally lasted longer than a year. It is, however, prudent to vaccinate every year, and to select for performing the operation a period when splenic fever has not yet become developed—in March and April. If the vaccinating is postponed until the fever is in the sheepfolds, there is the risk of attributing to vaccination the losses which in reality belong to the natural disease. Just as human vaccination cannot preserve from small-pox a patient who is already under the influence of small-pox, so the splenic vaccinations are powerless against a fever already in process of incubation.

It must not be assumed that the duration of immunity to animals after splenic vaccination cannot be compared with the duration of immunity from small-pox after Jennerian vaccination. Jenner and his contemporaries believed that vaccination was able to preserve from small-pox during the whole life. That illusion disappeared long ago, and now ten years has been fixed as the average duration of that immunity and of the interval which ought to separate successive vaccinations. This interval, moreover, is too long for a certain number of individuals. Besides, in order to judge of the immunity of antisplenic vaccination, we must not lose sight of the formidable trial which vaccinated animals have to undergo when inoculated with the most virulent virus. What doctor would dare to subject a vaccinated child to inoculation from virulent small-pox a year after its vaccination? Finally, taking into consideration the commercial and economic view of the life of a sheep—if such an expression may be used—the average scarcely exceeds three years. The duration, then, of the immunity that vaccination confers is about a third of the duration of the animal's life.