Louis Pasteur 8

Louis Pasteur 8

FOWL CHOLERA._ If fowls are naturally impervious to the infection of splenic fever, there is a disastrous malady to which they are subject, and which is commonly called 'fowl cholera.' Pasteur thus describes the disorder:--'The bird which is attacked by this disease is without strength, staggering, the wings drooping. The ruffled feathers of the body give it the shape of a ball. An overpowering somnolence takes possession of it. If forced to open its eyes, it appears as if it were awakened out of a deep sleep. Very soon the eyelids close again, and generally death comes without the animal changing its place, or without any struggle, except at times a slight movement of the wings for a few seconds.' The examination after death reveals considerable internal disorders. Here, again, the disease is produced by a microscopic organism. A veterinary surgeon of Alsace, M. Moritz by name, was the first who suspected the presence of microbes in this disease; a veterinary surgeon of Turin, M. Peroncito, depicted it in 1878; a professor of the veterinary school of Toulouse, M. Toussaint, recognised it, in his turn, in 1879, and sent to Pasteur the head of a cock which had died of the cholera. But, however skilful they were, these observers had not succeeded in deciding the question of parasitism. None of them had hit upon a suitable cultivating medium for the parasite, nor had they reared it in successive crops. This, however, is the only method of proving that the virulence belongs exclusively to a parasite. It is absolutely necessary, in the study of maladies caused by microscopic organisms, to procure a liquid where the infectious parasite can grow and multiply without possible mixture of other organisms of different kinds. An infusion of the muscles of the fowl, neutralised by potash, and rendered sterile by a temperature of 110 to 115 degrees, has proved to be wonderfully appropriate to the culture of the microbe of fowl cholera. The facility of its multiplication in this medium is almost miraculous. In some hours the clearest infusion begins to grow turbid, and is found to be filled with a multitude of little organisms of an extreme tenuity slightly strangulated at their centres. These organisms have no movement of their own. In some days they change into a multitude of isolated specks, so diminished in volume that the liquid, which had been turbid to the extent of resembling milk, becomes again almost as clear as at first. The microbe here described belongs to a totally different group from that of the vibrios. It is ranged under the genus called 'micrococci.' 'It is in this group,' said Pasteur on one occasion, 'that the microbes of the viruses which are yet unknown will probably be one day found.' In the cultivation of the microbe of fowl cholera, Pasteur tried one of the cultivating liquids which he had previously made use of with most success--the water of yeast--that is to say, a decoction of yeast in water rendered clear by filtration and then sterilised by a temperature of over 100 degrees. The most diverse microscopic organisms find in this liquid suitable nourishment, particularly if it has been neutralised. When, for example, the bacterium of splenic fever is sown in the liquid, it assumes in a few hours a surprising development. Now, it is remarkable that this medium is quite unsuited to the life of the microbe of fowl cholera. Not only does it not develop, but the microbe perishes in this liquid in less than forty-eight hours. May we not connect this singular fact with that which is observed when a microscopic organism proves innocuous in an animal which has been inoculated with it? It is innocuous because it cannot develop itself in the body of the animal, or because, its development being arrested, it cannot attain the vital organs. The decoction extracted from the muscles of the fowl is the only medium which really suits the microbe of fowl cholera. It suffices to inoculate the fowl with the hundredth, even the thousandth, part of a drop of this mixture, to produce the disease and cause death. But here is a strange peculiarity. If guinea-pigs are inoculated with this little parasite they are hardly ever killed by it. Guinea-pigs of a certain age generally exhibit only a local lesion at the point of inoculation, which ends in an abscess more or less prominent. After opening spontaneously, the abscess closes again and heals, while the animal preserves its appetite and its appearance of health. These abscesses sometimes last several weeks. They are surrounded by a pyogenic membrane and filled with a creamy pus, in which the microbe swarms side by side with the pus globules. It is the life of the microbe inoculated under the skin which causes the abscess. The abscess, with the membrane which surrounds it, becomes for the little organism a sort of closed vessel, which it is even easy to tap without sacrificing the guinea-pig. The organism is mixed with the pus in a state of great purity, and although it is localised its virulence is extreme. When fowls are inoculated with the contents of the abscess they die rapidly, while the guinea-pig, which has furnished the virus, gets well without the least suffering. A curious instance this is of the local evolution of a very virulent microscopic organism, which produces neither internal disorders nor the death of the animal upon which it lives and multiplies, but which can carry death to other species inoculated with it. Fowls and rabbits living among the guinea-pigs suffering from these abscesses might in a moment be smitten and perish, while the health of the guinea-pigs remained unchanged. To produce this result it would suffice that a little of the discharge from the abscess of a guinea-pig should get smeared over the food of the fowls and rabbits. An observer witnessing such deaths without apparent cause, and ignorant of this strange dependency, would no doubt be tempted to believe in the spontaneity of the disease. He would be far from supposing that the evil had originated in the guinea-pigs, which were all in good health. In the history of contagia what mysteries may some day be cleared up by even more simple solutions than this one! When some drops of the liquid containing this microbe are placed on the food of fowls, the disease penetrates by the intestinal canal. There the little organism increases in such great abundance that inoculation with the excrements of the injected fowls produces death. It is thus easy to account for the mode of propagation of this very serious disease, which depopulates sometimes all the poultry yards in the country. The only means of arresting the contagion is to isolate, for a few days only, the fowls and the chickens, to remove the dung heaps, to wash the yard thoroughly, especially with water acidulated with a little sulphuric acid, or carbolised water with two grammes of acid to the litre. These liquids readily destroy the microbe, or at least suspend its development. Thus all causes of contagion disappear, because, during their isolation, the animals already smitten die. The action of the disease, in fact, is very rapid. The repeated cultivation of the infectious microbe in the fowl infusion, passing always from one infusion to the next following, by sowing in the latter an infinitely small quantity, so to speak, of the virus--as much, for example, as may be retained on the point of a needle simply plunged into the cultivation--does not sensibly lessen the virulence of the microscopic organism. Its multiplication inside the bodies of fowls is quite as easy with the last as with the first culture. In short, whatever may be the number of the successive cultures of the microbe in the fowl infusion, the last culture is still very virulent. This proves the microbe to be the cause of the disease--a proof the same in kind as that which had already enabled Pasteur to show that splenic fever and septicæmia are produced by specific microbes. Like the bacillus of splenic fever, the microbe of the fowl cholera is an aerobic organism. It is cultivated in contact with the air, or in aerated liquids. At the same time, though it is entitled to be called an aerobic organism, it differs essentially in certain respects from the parasite of splenic fever. If splenic fever blood filled with filaments of the parasite be enclosed in a vessel protected from the air--say, in a tube closed at its two extremities--in a few days, eight or ten at the most, and much fewer in summer, the parasite disappears, or rather is reduced to fine amorphous granulations, and the blood loses all its virulence. If the same system of shutting out the air be employed with the blood of a fowl charged with the microbe of fowl cholera, this microbe will be preserved with its virulence for weeks, months, even years. Pasteur has been able to keep for three years tubes thus sealed, a drop of blood from which when cultivated in fowl infusion, sufficed to infect the birds in the poultry yard with cholera. And not only is the microbe preserved thus in the blood contained in the tube; the same occurs if fowl infusion be put into tubes and then sealed by the flame of a lamp. When, in course of time, such tubes lose their virulence, it is because the vitality of the organism is extinct. The moment the contents of the tube cease to be virulent, it is a sign that the contagium is dead. It is useless, then, to attempt to cultivate it: the microbe cannot be revived. Here, then, is a third virulent disease, also produced by a microscopic organism. The characteristics of fowl cholera are very different from those of splenic fever and acute septicemia, and these three microbes do not in the least resemble each other. But, glancing back over Pasteur's work, are not the diseases of silkworms, pebrine and flacherie, also virulent diseases? Thus, in so many things, through so many studies, the same connection holds good. Each discovery of Pasteur's is linked to those which precede it, and is the rigorous verification by experimental method of a preconceived idea. 'Nothing can be done,' said he one day, 'without preconceived ideas; only there must be the wisdom not to accept their deductions beyond what experiments confirm. Preconceived ideas, subjected to the severe control of experimentation, are the vivifying flame of scientific observation, whilst fixed ideas are its danger. Do you remember the fine saying of Bossuet? "The greatest sign of an ill-regulated mind is to believe things because you wish them to be so." To choose a road, to stop habitually and to ask whether you have not gone astray, that is the true method.' It is this method which conducted him in 1880 to that wonderful discovery, the attenuation of contagia. What certain of these contagia are, we have already seen. We shall now learn what they become in the hands of Pasteur.      _THE ATTENUATED VIRUS, OR VACCINATION OF VIRULENT DISEASES._                      THE VACCINE OF FOWL CHOLERA. Among the scourges which afflict humanity there are none greater than virulent diseases. Measles, scarlatina, diphtheria, small-pox, syphilis, splenic fever, yellow fever, camp typhus, the plague of the East--what a terrible enumeration! I pass over some, such as glanders, leprosy, and hydrophobia. The history of these diseases presents extraordinary circumstances. The most strange, assuredly, is that which has been from all time established with a great number of these diseases, that they are non-recurrent. As a general rule, notwithstanding some rare exceptions, man can only have measles, scarlet fever, plague, yellow fever once. What explanation, even hypothetically, can be given of such a fact? Still more difficult is it to explain how vaccination, which is itself a virulent though benign disease, preserves from a more serious malady, the small-pox? Has there ever been a discovery more mysterious in its causes and origin, standing, as it does, alone in the history of medicine, and for more than a century defying all comparison? After dwelling long on Jenner's discovery this question arose in Pasteur's mind: If contagious maladies do not repeat themselves, why should there not be found for each of them a disease different from them, but having some likeness to them, which, acting upon them as cow-pox does upon small-pox, would have the virtue of a prophylactic? A chance occurrence, one of those chances which not unfrequently occur to those who are steadfastly looking out for them, opened out to Pasteur the way to a discovery which may well be called one of the greatest discoveries of the age. In causing the microbe of fowl cholera to pass from culture to culture, in an artificial medium, a sufficient number of times to render it impossible that the least trace of the virulent matter from which it originally started should still exist in the last cultivation, Pasteur gave in an absolute manner the proof that infectious microbes are the sole authors of the diseases which correspond to them. This culture may be repeated ten, twenty, a hundred, even a thousand times: in the latest culture the virulence is not extinguished, or even sensibly weakened. But it is a fact worthy of attention that the preservation of the virulence in successive cultures is assured only when no great interval has been allowed to elapse between the cultures. For example, the second culture must be sown twenty-four hours after the first, the third twenty-four hours after the second, the hundredth twenty-four hours after the ninety-ninth, and so on. If a culture is not passed on to the following one until after an interval of several days or several weeks, and particularly if several months have elapsed, a great change may then be observed in the virulence. This change, which generally varies with the duration of the interval, shows itself by the weakening of the power of the contagium. If the successive cultures of fowl cholera, made at short intervals, have such virulence that ten or twenty inoculated birds perish in the space of twenty-four or forty-eight hours, a culture which has remained, say, for three months in its flask, the mouth of which has been protected from the introduction of all foreign germs by a stopper of cotton wool, which allows nothing but pure air to pass through it--this culture, if used to inoculate twenty fowls, though it may render them more or less ill, does not cause death in any of them. After some days of fever they recover both their appetite and spirits. But if this phenomenon is extraordinary, here is one which is surely in a different sense singular. If after the cure of these twenty birds they are reinoculated with a very virulent virus--that, for instance, which was just now mentioned as capable of killing its hundred per cent. of those inoculated with it, in twenty-four or forty-eight hours--these fowls would perhaps become rather ill, but they would not die. The conclusion is simple; the disease can protect _from_ itself. It has evidently that characteristic of all virulent diseases, that it cannot attack a second time. However curious it may be, this characteristic is not a thing unknown in pathology. Formerly it was the custom to inoculate with small-pox to preserve from small-pox. Sheep are still inoculated to preserve them from the _rot_; to protect horned cattle from peripneumonia they are inoculated with the virus of the disease. Fowl cholera offers the same immunity; it is an additional scientific acquisition, but not a novelty in principle. The great novelty which is the outcome of the preceding facts, and which gives them a distinct place in our knowledge of virulent diseases, is that we have here to do with a disease of which the virulent agent is a microscopic parasite, a living organism cultivated outside of the animal body, and that the attenuation of the virulence is in the power of the experimenter. He creates it, he diminishes it, he does what he wishes with it; and all these variable virulences he obtains from the maximum virulence by manipulation in the laboratory. Looked at in juxtaposition with the great fact of vaccination for small-pox, this weakened microbe, which does not cause death, behaves like a real vaccine relatively to the microbe which kills, producing a malady which may be called benign, since it does not cause death, but is a protection from the same malady in its more deadly form. But for this enfeebled microbe to be a real vaccine, comparable to that of cow-pox, must it not be fixed, so to speak, in its own variety, so that there should be no necessity for having recourse again to the preparation from which it was originally derived? Jenner, when he had demonstrated that cow-pox vaccination preserved from small-pox, feared for some time that it would be always necessary to have recourse to the cow to procure fresh vaccinating matter. His true discovery consisted in establishing that the cow-pox from the cow could be dispensed with, and that inoculation could be performed from arm to arm. Pasteur made his enfeebled microbe pass from one cultivation to another. What would it become? Would it resume its very active virulence, or would it preserve its moderate virulence? The virulence remained enfeebled and, we may say, unchanged. This showed it to be a real vaccine. Some veterinary surgeons and farmers, on the announcement of this discovery, applied to Pasteur for a vaccine against the disease which was so disastrous among their poultry. Some trials were made, and all succeeded beyond expectation. To preserve this vaccine it must be secured from contact with the air, the cultures being enclosed in tubes, the extremities of which are sealed by the flame of a blowpipe. What takes place during that interval of time intentionally placed between two successive cultivations of the cholera microbe--that interval which is employed in effecting the attenuation and producing the vaccine? What is the secret of this result? The agent which intervenes is no other than the oxygen of the air. Here is the proof. If the cultivation of this microbe is carried on in a tube containing very little air, and if the tube is then closed by the flame of a lamp, the microbe, by its development and life, quickly appropriates all the free oxygen contained in the tube, as well as the oxygen dissolved in the liquid. Thus, completely protected from contact with oxygen, the microbe does not become sensibly weakened for months, sometimes even for years. The oxygen of the air, then, appears to be the cause of modification in the virulence of the microbe. But how, then, is the absence of influence on the part of the atmospheric oxygen, in the successive cultures which are practised every twenty-four hours, to be explained? There is, in Pasteur's opinion, but one possible explanation; it is that the oxygen of the air in this latter case is solely employed in the life of the microbe. A culture has a duration of some days; in twenty-four hours it is not terminated. The air which comes in contact with it is then entirely employed in nourishing and largely reproducing the microbe. During the longer intervals of culture, the air acts only as a modifier, and at last there arrives a moment when the virulence is so much weakened as to become nil. This very extraordinary fact is, then, established that the virulence may be entirely gone while yet the microbe lives. The cultures offer the spectacle of a microbe indefinitely cultivable, yet, on the other hand, incapable of living in the bodies of fowls, and in consequence deprived of virulence. May not this domesticated microbe, as M. Bouley calls it, be compared to those inoffensive microbes of which there are so many in nature? May not our common microbes be those organisms which have lost their former virulence? But may not these harmless microbes, become infectious in some particular circumstances? And if there are fewer virulent maladies now than there were in times past, might not the number of these maladies again increase? Questions multiply as the facts relating to the attenuation of a virus suggest inductions, awaken ideas, and throw new lights upon a problem which, until within these last few years, has remained so obscure. Formerly it was believed that these viruses were morbid entities. A virus was a unity. This opinion has still its declared upholders. According to Pasteur a virus has different degrees of virulence; it can pass from the weakest virulence to the maximum. Modifying, at will, the virus of fowl cholera, Pasteur inoculates some hens, for instance, with a virus too attenuated to protect from death, but which nevertheless is effectual in securing them against a virus stronger than itself. The second virus will preserve them from the attacks of a third virus, and thus passing from virus to virus they end by being guaranteed against the most deadly virulences. The whole question of vaccination resolves itself into knowing at what moment a certain degree of virus attenuation is a guarantee of protection against the mortal virus. It seems that between small-pox and cow-pox facts of a similar kind take place. It is probable that vaccination rarely gives perfect security against the infection of a very malignant small-pox; moreover, during epidemics of small-pox many persons who have been previously vaccinated are attacked, and some even die of the disease. As regards the practice of vaccinating fowls against the cholera peculiar to them--which, though it certainly is not of the same importance as human vaccination, is a scientifically _capital_ fact--we may hope that whatever the differences of receptivity in different races, or in different individuals of the same race, there will be found vaccines to suit them all, special care being taken to resort to the employment of two successive vaccines of unequal power, employed after an interval of ten or fifteen days. The first vaccine may always be chosen of a degree of weakness which will not in any case cause death, and yet of sufficient strength to prevent dangerous consequences from the second vaccine, which would in some cases be fatal if employed at once, and to enable it to act as a vaccine against the most virulent virus. With regard to the preparation of vaccines, and the ascertaining of their proper strength, it is necessary to make trials upon a certain number of fowls, even at the risk of sacrificing a few in these preliminary experiments. Beyond such questions of manipulation there remains still a scientific question. How are the effects of vaccination to be conceived? What explanation can be given of the fact that a benign disease can preserve from a more serious and deadly one? Pasteur long sought for the solution of this problem. Without flattering himself that he has unravelled the difficulty, he has nevertheless amassed facts which, amid these physiological mysteries, permit us to frame a hypothesis which can satisfy the mind. Pasteur believes, for example, that the vaccine, when cultivated in the body of the animal, robs the globules of the blood, for example, of certain material principles which the vital actions take a long time to restore to the system, and which to the most deadly contagium is a condition of life. The impossibility of action of the progressive virus and of the deadly virus is thus accounted for. When Pasteur communicated to the Academy of Sciences these important and unforeseen facts, they were at first received with hesitation. It was not without some surprise that the word vaccination, hitherto exclusively reserved for Jenner's discovery, was heard applied to fowl cholera. At the International Medical Congress held in London in August 1881, Pasteur, in the presence of 3,000 doctors of medicine from all parts of the world, who received him with an enthusiasm which reflected glory on France, justified the name that he had given to his prophylactic experiments. 'I have lent,' he said, 'to the expression vaccination an extension that I hope science will consecrate, as a homage to the merit and immense services rendered to humanity by one of the greatest men of England--Jenner.' Still, while rendering homage to the sentiment which induced Pasteur to efface himself in favour of Jenner, we may be permitted to say that there is no likeness between the two discoveries. Great as was the discovery of Jenner, it was but a chance observation, which had no ulterior development; and for a whole century, medicine has not been able to derive anything from it beyond its actual application, which is the one result achieved. 'Vaccination is vaccination,' an opponent of Pasteur's, who was driven hard, was obliged to say. The opponent found no other answer, and he could not have found any other. The cow-pox is a malady belonging exclusively to a race of animals. Man can only observe it; he cannot produce it. Suppress cow-pox and there will be no more vaccination. In the French discovery, on the contrary, it is the deadly virus itself which serves as a starting point for the vaccine. It is the hand of man which makes the vaccine, and this vaccine may be artificially prepared in the laboratory, in sufficient quantity to supply all needs. What a future is presented to the mind in the thought that the virus and its vaccines are a living species, and that in this species there are all sorts of varieties susceptible of being fixed by artificial cultivation! The genius of Jenner made a discovery, but Pasteur discovered a method of genius. 'This is but a beginning,' said M. Bouley on the day when Pasteur announced these facts to the Academy of Sciences. 'A new doctrine opens itself in medicine, and this doctrine appears to me powerful and luminous. A great future is preparing; I wait for it with the confidence of a believer and with the zeal of an enthusiast.'                     _THE VACCINE OF SPLENIC FEVER._ 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 Ecole 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.                       _THE RETURN TO VIRULENCE._ After having reduced the microbes of fowl cholera and splenic fever to all degrees of virulence, and brought them to a point where they could no longer multiply in the bodies of animals inoculated with them, and fixed them in media appropriate to their life, Pasteur asked himself whether it would not be possible to restore to these attenuated microbes--weakened to such a degree as to have lost all virulence--a deadly virulence, and to render them again capable of living and multiplying in the bodies of animals. Experiment soon confirmed this mental prevision. An attenuated splenic fever virus which could cause no danger of disease or death to guinea-pigs of a year, or a month, or even a week old, could kill a little guinea-pig just born, or one or two days old. The attenuated microbe could multiply itself in the blood of so young an animal. We can well imagine that in an animal, scarcely formed, the power of oxygenation of the blood globules is not as yet capable of preventing the aerobic microbe from turning to its own account the oxygen of the blood. The disease does its work and death supervenes. After all, there is nothing surprising in the fact that the vital resistance of a newly-born guinea-pig should differ from that of an adult one. But what is very remarkable is, that if an older guinea-pig be inoculated with the blood of one a day old; if a third, still older, be inoculated with the blood of the second; and so on; the virulence of the microbe will be gradually reinforced--that is to say, the usual habit of this parasite to develop itself in the body of the animal will be restored. The process may be likened to that of an animal or vegetable species, passing by successive stages and long sojourns, from one region to another very distant one, subjected to quite new conditions of climate, and gradually becoming acclimatised to the last one. How great, then, must be the importance of the medium of cultivation, with regard to the virulence of the microbes of communicable diseases! Cultivating the microbe by passing it from one guinea-pig to another, we soon arrive at a strength capable of killing guinea-pigs of a week, a month, or several years old, until at last the smallest drop of the blood of these guinea-pigs suffices to kill a sheep; and from the sheep we may pass on to the ox. The same is the case with the microbe of fowl cholera. When it has ceased to have any effect upon fowls, its virulence can be restored by inoculating small birds. Blackbirds, canaries, sparrows, all die, if the virus has not been too much attenuated; and the effect is similar on young chicks. Thus by several successive transitions from bird to bird a virulence may be fostered capable of destroying full-grown fowls. These facts suggested to Pasteur certain inductions which may be well founded. Is not the attenuation of the virus by the influence of the air one of the factors in the extinction of great epidemics? And may not the reappearance of these scourges be accounted for by the reinforcement of the virulence?