II.
In these two virulent maladies, then, splenic fever and septicæmia, the researches of Pasteur had clearly established the parasitic theory. A grand and novel opening was made for future studies on the origin of diseases. Yet, judging from the surprising differences which separate septicæmia and splenic fever, we can foresee that should the future, copying the past, in regard to this and still more recent discoveries, have in store, as it no doubt has, the knowledge of new microbes of disease, the specific properties of these microscopic organisms will demand, for each new exploration, ceaselessly repeated efforts, not only to make the existence of these organisms evident, but also to furnish decisive proofs of their morbific power. But the question which may be considered as already solved is the non-spontaneity of these infectious microbes. By what is called spontaneous disease is meant parasitic disease. But in the present state of science spontaneous disease has no more existence than spontaneous generation. Such aphorisms, however, are not allowed to pass without occasional contradictions, all the more vehement from their rarity. At the International Medical Congress held in London, August 1881, Dr. Bastian, who practises in one of the principal hospitals of London, declared that though he was unable to deny the existence of parasitic diseases, yet, in his opinion, the microbes were the effect and not the cause of these diseases.
'Is it possible,' cried Pasteur, who was present at the meeting, 'that at this day such a scientific heresy should be held? My answer to Dr. Bastian will be short. Take the limb of an animal and crush it in a mortar; let there be diffused in this limb, around these crushed bones, as much blood, or any other normal or abnormal liquid as you please. Take care only that the skin of the limb is neither torn nor laid open, and I defy you to exhibit on the following day, or during all the time the malady lasts, the least microscopic organism in the humours of this limb.'
After the example of Liebig in 1870, Dr. Bastian did not accept the challenge.
But if a disease like splenic fever is carried by a microbe, this microbe is under the influence of the medium in which it finds itself. It does not develop everywhere. Easily inoculable and fatal to the ox, the sheep, the rabbit, and the guinea pig, splenic fever is very rare in the dog and in the pig. These must be inoculated several times before they contract the disease, and even then it is not always possible to produce it. Again, there are some creatures which are never assailable by it. It can never be taken by fowls. In vain they are inoculated with a considerable quantity of splenic blood; it has no effect upon them. This invulnerability had very much struck Pasteur and his two assistants, Joubert and Chamberland. What was it in the body of a fowl that enabled it to thus resist inoculations of which the most infinitesimal quantity sufficed to kill an ox? They proved by a series of experiments that the microbe of splenic fever does not develop when subjected to a temperature of 44° Centigrade. Now, the temperature of birds being between 41 and 42 degrees, may it not be, said Pasteur, that the fowls are protected from the disease because their blood is too warm—not far removed from the temperature at which the splenic fever organism can no longer be cultivated? Might not the vital resistance encountered in the living fowl suffice to bridge over the small gap between 41-42, and 44-45 degrees? For we must always allow for a certain resistance in all living creatures to disease and death. No doubt, life to a parasite in the body of an animal would not be as easy as in a cultivating liquid contained in a glass vessel. If the inoculating microbe is aerobic, it can only be cultivated in blood by taking away the oxygen from the globules, which retain it with a certain force for their own life. Nothing was more legitimate than to suppose that the globules of the blood of the fowl had such an avidity for oxygen that the filaments of the splenic parasite were deprived of it, and that their multiplication was thus rendered impossible. This idea conducted Pasteur and his assistants to new researches. 'If the blood of a fowl was cooled,' they asked, 'could not the splenic fever parasite live in this blood?'
The experiment was made. A hen was taken, and, after inoculating it with splenic fever blood, it was placed with its feet in water at 25 degrees. The temperature of the blood of the hen went down to 37 or 38 degrees. At the end of twenty-four hours the hen was dead, and all its blood was filled with splenic fever bacteria.
But if it was possible to render a fowl assailable by splenic fever simply by lowering its temperature, is it not also possible to restore to health a fowl so inoculated by warming it up again? A hen was inoculated, subjected, like the first, to the cold-water treatment, and when it became evident that the fever was at its height it was taken out of the water, wrapped carefully in cotton wool, and placed in an oven at a temperature of 35 degrees. Little by little its strength returned; it shook itself, settled itself again, and in a few hours was fully restored to health. The microbe had disappeared. Hens killed after having been thus saved, no longer showed the slightest trace of splenic organisms.
How great is the light which these facts throw upon the phenomenon of life in its relation to external physical conditions, and what important inferences do they warrant as to the influence of external media and conditions upon the life and development of living contagia! There have been great discussions in Germany and France upon a mode of treatment in typhoid fever, which consists in cooling the body of the patient by frequently repeated baths. The possible good effects of this treatment may be understood when viewed in conjunction with the foregoing experiment on fowls. In typhoid fever the cold arrests the fermentation, which may be regarded as at once the expression and the cause of the disease, just as, by an inverse process, the heat of the body arrests the development of the splenic fever microbe in the hen.
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, pébrine 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 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.
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?
'The accounts which I have read,' Pasteur remarked some months ago, 'of the spontaneous appearance of the plague in Benghazi in 1856 and in 1858 tend to prove that this outbreak could not be traced to any original contagion. Let us suppose, guided by the facts now known to us, that the plague, a malignant disease belonging to certain countries, has germs of long duration. In all these countries its attenuated virus must exist, ready to resume its active form whenever the conditions of climate, of famine, of misery present themselves afresh. The condition of long duration in the vitality of the germs of evil is not even indispensable; for, if I may believe the doctors who have visited these countries, in all places subject to the plague, and in the intervals of the great outbreaks of the epidemic, cases may be met with of people attacked with boils, not fatal, but resembling those of the deadly plague. Is it not probable that these boils contain an attenuated virus of the plague, and that the passage of this virus into exhausted bodies, which abound only too freely in periods of famine, may restore to it a greater virulence?
'The same may be the case with other maladies which appear suddenly, like typhus in armies and in camps. Without doubt, the germs which are the authors of these diseases are everywhere scattered around, but attenuated; and in this state a man may carry them about him or in his intestinal canal without great damage. They only become dangerous when, by conditions of overcrowding, and perhaps of successive developments on the surfaces of wounds, in bodies enfeebled by disease, their virulence is reinforced.'
ETIOLOGY OF SPLENIC FEVER.
M. Pasteur had triumphed over splenic fever with as much rigour as precision. But he considered that he had still to make one further investigation. He had established the effects of the pest; he had discovered a preventive method with which to combat it: he now wished to know the origin of the evil. Whence comes splenic fever? Why is it endemic in certain departments of France, in certain parts of Russia, Germany, Austria, Italy, Spain, and America? How is it sustained? It was for a long time believed that splenic fever was born spontaneously under the influence of various accidental causes. M. Bouley has related, in his learned work on the 'Progress of Medicine by Experimentation,' that in 1842 the Minister of Agriculture, M. Cunin-Gridaine, at the request of the deputies of the departments that were ravaged by the epidemic, entrusted to M. Delafond, a professor of the school at Alfort, the task of investigating this malady, commonly called the 'blood disease,' in the districts in which it was raging. He was to search out its causes, and ascertain whether they did not result from the system of cultivation prevalent in the country.
M. Delafond arrived in Beauce. One fact struck him—namely, that almost all the animals attacked by the disease were young, fine, and vigorous: those, in short, that gave the best promise. Viewing the richness of the soil and the abundance and quality of the crops in conjunction with this observation, Delafond at once elaborated a speculative theory. 'The blood disease,' said he, 'is nothing but an overfulness—an excess of blood circulating in the vessels, and especially the predominance in that liquid of red globules.'
Starting from this idea, his one object was, by means of logical deduction, to trace everything to this fundamental error. He analysed the soil, and demonstrated to what extent it was fitted to furnish crops that were rich and abounding in nutritive properties. He analysed the plants. He then complacently referred the richness of blood of the Beauce sheep to the richness in nitrogenous principles of the substances on which they were fed. He examined the lesions of the diseased animals, and concluded that they were the consequence of the blood containing too large a proportion of the organic elements, called globules, fibrine, albumen, and too small a proportion of water.
'Reduce the proportion of nutritious elements,' he wrote as his advice to the agriculturists, 'mix roots with all that is too rich in nitrogenous principles, and you will reduce in proportion the losses caused by the excess of the ultra-nutritious substances with which you supersaturate your cattle.'
'Such is the very logical conclusion to which Delafond was led,' adds M. Bouley, ridiculing these observations, based on a method of reasoning, instead of on the experimental method. 'And as a fresh proof of his theory he mentions the fact that the disease decreases as you descend the country towards the Loire. On the right bank of that river—in Sologne, for instance, which is a low, sandy, damp district—blood disease is unknown. In the arrondissements of Gien and Montargis and in parts of those of Orleans and Pithiviers it prevails but little. There, Delafond imperturbably remarks, the soil is sandy and the herbage not nearly so rich as in the Beauce plateau; and there the blood disease is consequently less common.'
When we consider that such opinions could be written unchallenged only forty years ago, that they could even borrow a scientific character from the inspiration that gave them birth, we can see the progress that has since been made, and can realise how great were the obscurity and uncertainty which have been dispelled by the experimental method.
The presence of a parasite having been brought to public notice in the blood of animals suffering from splenic fever, at the very time when Pasteur had shaken the belief in spontaneous generation, people grew accustomed to the idea that the stricken animals might have contracted the germs of the malady from the outer world, without any spontaneous birth, strictly so called, of the disease. This opinion was strengthened by a knowledge of the spores of the splenic bacillus. Pasteur, aided by Messieurs Chamberland and Roux, commenced experiments with a view to solving this difficult etiological question. The first experiments took place in the fields of a farm at the village of St. Germain, near Chartres. Several groups of sheep were fed on lucern grass which had been sprinkled with artificially-reared splenic fever bacteria, or with their germs or spores. Although all the sheep of the same group absorbed an immense number of the spores of the parasite, many survived, even after being visibly affected. Those that died showed all the symptoms of what is called spontaneous splenic fever. The period of incubation lasted as long as eight or ten days, although, in its latter stages, the disease exhibited those startling features which have caused a belief that the incubating period is a very short one—short, that is to say, for those conditions of contagion where the parasite is not deposited in its pure state under the animal's skin.
But if prickly plants (notably the pointed ends of dried thistle leaves, or beards of barley blades cut into little bits about a centimeter in length) were added to this infected food, the mortality increased to a striking extent. On examination after death, the lesions of these animals were found to be similar to those observed in sheep which were attacked by splenic fever in sheds, or which died of the disease in the open fields.
From that time forward, the idea which had been predominant in the minds of Pasteur and his fellow-workers during all their inquiries, was materially strengthened. They were convinced that the animals which died of blood disease in the department of Eure et Loire had been infected by germs or spores of the splenic microbe contained in their food; but the question remained, Whence came these germs?
From the moment when all belief in the spontaneous generation of the parasite is rejected, attention is naturally drawn to the possible consequences which may arise from burying in the earth animals which have died of splenic fever. In the greater number of cases, when the knacker's establishment is too far off and the dead animal is of little value, a trench is dug on the spot, at a depth varying from half a meter to a meter. If the animal dies in a field, it is buried where it falls; if it dies in a shed the body is carried into a neighbouring field. There it is buried, and putrefaction sets in; and since all the splenic fever filaments of the blood are destroyed by putrefaction, it was thought that no dissemination of the germs of splenic fever, after the animal had been buried, could occur. Pasteur showed that this opinion rested on a superficial observation. Even when the animal is not cut up, blood spreads itself outside of the body in more or less abundance. Is it not an habitual characteristic of the disease, that at the time of death blood issues from the nostrils and the mouth, and that the urine is often bloody? All around the corpse, therefore, the earth is polluted with blood. Moreover it takes several days for the splenic fever microbe to resolve itself into harmless granulations by the action of gases, other than oxygen, which putrefaction generates. During this time, the excessive inflation of the dead body causes the liquids of the interior to issue from all the natural apertures. How often also, a rent in the skin or the tissues increases this flow. The blood and other matters, mixed with the surrounding aerated soil, are no longer in the conditions of putrefaction, but rather in those which form a suitable medium of cultivation for the microbe. Experiments confirmed these views. Adding some splenic fever blood to earth sprinkled with the water of yeast, or with urine, at summer temperature, or at the temperature which the fermentation of a dead body keeps up around it, as in a dung heap, in less than twenty-four hours the splenic fever filaments deposited with the blood had multiplied and resolved themselves into spores. These spores were afterwards found in their state of latent life, ready to germinate and to communicate splenic fever, after remaining in the earth for months, and even years.
These experiments, curious as they were, were only, so to speak, laboratory experiments. It was necessary to investigate what happened in the open country with all the variations of dryness, of damp, and of cultivation. A happy inspiration came to Pasteur and his assistants. They had buried in the midst of summer, in an isolated corner of the farm of St. Germain, near Chartres, a sheep which had died of natural splenic fever, and of which they had made the autopsy. Ten months afterwards, and again fourteen months afterwards, the idea occurred to them of collecting some of the earth from this grave. After having examined it, and established the presence of the spores of the microbe, they produced, by the inoculation of guinea-pigs, the splenic disease and death. But the circumstance which deserves the greatest attention, is that the same experiment was successfully made with the earth on the surface of the grave, though this earth had not been disturbed during the interval. Some experiments were afterwards made on the earth of some trenches dug in a meadow of the Jura, where some cows which had died of splenic fever had been buried at a depth of two meters. Two years afterwards, by successive washings of the earth on the surface of the graves, deposits were extracted which at once produced splenic disease. At three trials within these two years the same surface earths produced splenic fever, while, away from the graves, the earth exhibited nothing of the kind. Finally, Pasteur and his assistants proved that on the surface of the earth which covered the buried animals, the germs were again found, after all the operations of ploughing, sowing, and reaping.
But how, it will be asked, can the earth, which is so powerful a filter, allow the germs of microscopic organisms to rise again to its surface? Is one not tempted here to quote Pasteur against himself, since, in his joint researches with M. Joubert, Pasteur had proved that the waters of springs issuing from the earth, even at a shallow depth, are entirely free from germs? Such waters, nevertheless, being supplied from the earth's surface, which is constantly washed by rain, the effect must be to carry down the finest particles to the springs. But these latter, notwithstanding conditions so conducive to their pollution, remain perfectly pure. Can there be a better proof that earth of a certain thickness will arrest all solid particles, even the most minute? Nevertheless, in these experiments on splenic fever, we hear of microscopic germs, starting from the depths and coming up to the surface—that is to say, in a direction contrary to the flow of the rain. This is an enigma.
The explanation will cause surprise. The earth-worms transport the germs, and bring up, from the depths where they lie buried, the terrible microbes. In the tiny cylinders of earth which the worms deposit on the surface of the soil, after the dews of the morning or after rain, the splenic germs are to be found. It is easy to prove this directly. If in earth, with which spores of the microbe have been previously mingled, we place some worms, and at the end of several days open the bodies of these worms, with all necessary precautions, so as to extract from them the earthy matter which fills their intestinal canals, we find in them large numbers of splenic fever spores. It is, then, absolutely proved, that if splenic fever germs exist, as they often do, in the light earth which covers the pits in which animals dead of that disease lie buried, these germs result from the disintegration by rain of the little excremental cylinders deposited by the earth-worms. The dust of this disintegrated earth spreads itself over the grasses on a level with the soil, and thus it is that animals come to find on the pasture-field, and in particular kinds of forage, the germs of splenic fever by which they are infected.
'In these results,' said Pasteur a short time ago at the Academy of Medicine, 'what outlooks are opened to the mind in regard to the possible influence of earths in the etiology of diseases, and the possible danger of the earth of cemeteries!'
The earth-worms also bring to the surface other germs, which, while they are as harmless to the worms as the splenic germs, are nevertheless bearers of diseases to which animals are liable. All sorts of germs are found in them, and the germs of splenic fever are in fact always associated with those of putrefaction and septicæmia.
'And now,' concluded Pasteur, when laying before the Academy a rapid survey of the etiology of splenic fever, 'is not the remedy naturally indicated? We should never bury animals in fields destined either for cultivation, for forage, or for sheep pasture. When it is possible a sandy soil should be chosen for the purpose, or any poor calcareous soil, dry, and easily desiccated—in a word, soil not suited to the existence of earth-worms.' M. Tisserand, Director of Agriculture, has remarked that splenic fever is unknown in the region of the Savarts of Champagne, although it is surrounded by countries invaded by the disease. If the conditions of commerce introduce splenic fever, it is but a passing accident. Must not this be attributed to the fact that in these poor soils, such as that of the camp at Châlons, where the thickness of arable soil is only from 4 to 5 inches, superposed upon chalk, the worms cannot live? In such a soil the burial of a splenic fever animal will give rise to great quantities of germs, which, owing to the absence of earth-worms, will abide in the depths of the soil and remain harmless. Finally, it has been proved that the countries subject to splenic fever have an argillaceous-calcareous soil, and that the disease is unknown in schistose and granitic soils. The contrast of the results, in relation to such differences of soil, is seen sometimes in the Department of the Aveyron, between the right and left side of one and the same road or watercourse.
May we not now in all confidence assert that, if the cultivators choose, splenic fever may soon be a thing of the past among their animals, their shepherds, and among the butchers and the tanners of the towns, because splenic fever and malignant pustule are never spontaneous? The disease exists only where it has been sown, or where it has been diffused by the unconscious instrumentality of the earth-worm.
The progress of vaccination will also contribute to the disappearance of splenic fever; for this preventive, if extensively used, as there is no doubt it will be, must end by establishing a race of domestic animals which, having all sprung from vaccinated parents, will in consequence be more resistant to the disease in its worst form. It will be with them in relation to splenic fever as it is with ourselves in relation to small-pox. It is a well-known fact that the ravages of small-pox are much less considerable in our days than when it first appeared in Europe. It is difficult not to attribute this, at least in part, to the prevalence of vaccination.
In the populations where small-pox is introduced for the first time it has an exceptional intensity. Some months ago a significant fact of this nature occurred in Paris. A whole family of Esquimaux perished from small-pox in the 'Jardin d'Acclimatation.' They had never been vaccinated, nor had their ancestors. They were new to the attacks of small-pox, which did not spread beyond them.
METHOD OF DISCUSSION AND CONTRADICTIONS.
Every new discovery produces a revolution in general ideas; a revolution gladly hailed by some, but opposed by others as disturbing their habits of thought and reasoning. Those also who are thrown out in their calculations, while engaged in working out a problem in any way similar to the one that has been solved, too often atone for their dilatoriness by furious denial of the newly asserted truth. The great fact of the attenuation of virus, the artificial production of the vaccines of chicken cholera and of splenic fever, the importance of their employment for the preservation of animals from these diseases, excited throughout the world a surprise and enthusiasm which passionate critics soon sought to disparage. The fiercest attack was from Germany. It commenced immediately after Pasteur's triumph at the International Congress of Medicine held in London in 1881. The German doctor Koch and his colleagues, MM. Gaffki and Lœffler, published in Berlin, in the report of the German Sanitary Office, a kind of scientific tirade against the discovery of virus vaccine, and the possibility of utilising it in the large operations of cattle-breeding.
At the London Congress Dr. Koch had said to a French physician that the possibility of attenuating virus was a thing too good to be true. The whole question was therefore reopened by Dr. Koch and his disciples. At first Pasteur let the torrent flow; but, not being the man to give way before an adversary, he at last declared that the attacks of the German savants must be repelled at Berlin itself. Continual applications for splenic vaccine were made to him from different parts of Germany. M. Pasteur replied that, seeing that the discovery was so formally contested in Prussia, it would be well, before sending any vaccine abroad, to institute a great demonstrative experiment, as had been done at Pouilly-le-Fort.
Dr. Roloff, head of the Veterinary School of Berlin, hastened to take the initiative, by an application to the German Minister of Agriculture. The minister at once nominated a Commission to follow the experiments in vaccination and to draw up a report for the German Government. M. Pasteur entrusted the conduct of the vaccinations to his new colleague, Louis Thuillier, who accepted with deep and silent joy the management of an experiment that was to test a French discovery. He was always ready for anything, this brave Thuillier, who was destined to die, a martyr to the cause of science, in the full promise of his youth, and in the full hope of glory. His courage and his work were alike great and silent. In the laboratory he would spend days, even weeks, without speaking, bent over his microscope with tenacious resolution, endeavouring to follow Pasteur in all his investigations: proud to live near his illustrious master, happy to be his disciple and to be loved by him almost as a son. What a vacancy he has left in the laboratory! What a place he might have held in science!
The composition of the German Commission, over which M. Beyer, member of the Superior Council of Government, presided, showed clearly the importance attached by Germany to the investigation of this French discovery. Among its members was the famous Professor Virchow.
The experiments were carried out on the estate of Pakisch. The minutes and reports of the Commission left no doubt as to the correctness of the facts announced by Pasteur. But, as the negations of Dr. Koch and his colleagues embraced questions beyond that of the prophylaxy of splenic fever, Pasteur did not rest content with this initial success; he sought for a fresh opportunity of convincing his opponents. This opportunity occurred in September 1882, when an International Hygienic Congress was held at Geneva. Thither went Pasteur, hoping to meet Dr. Koch at the sittings; and he was not disappointed. Dr. Koch was there, surrounded by his disciples. From the tribune of the Congress, Pasteur refuted his criticism, exposed his errors, and challenged him to a discussion in the presence of competent judges. There was an instantaneous salvo of applause, and everyone awaited Dr. Koch's reply. But he declined all debate, reserving his case for careful and deliberate statement in the press.
It took three months for Dr. Koch to bring out a small pamphlet, and these three months had borne their fruit. The discovery of the attenuation of virus, which had been so vehemently attacked only a year before in the report of the Sanitary Office, was now extolled by Dr. Koch as a discovery of the first importance. Being, however, unwilling absolutely to stultify himself, he continued the attack by denying its efficacy in practical agriculture.
The clear, direct style of argument, which goes straight to its point, was invariably adopted by Pasteur.
'Contradictions may retard, although they cannot ultimately prevent, the recognition of truth,' he once remarked to me when walking in the gardens of the École Normale; 'that is why it is so important to remove the obstacles which temporarily clog and hamper it. In scientific discussions, it is not as in politics,' he added with a smile, 'where demonstration is often difficult. In the natural sciences, doctrines must be based on an assemblage of results, of observations, and of experiments. If a doctrine is challenged, it seldom happens that its truth or falsehood cannot be established by the application of some crucial test. Even a single experiment will often suffice either to refute or consolidate the doctrine.'
Reviewing the labours of the past forty years, Pasteur then called to mind the numerous controversies in which he had been engaged. Not only had he been attacked by Pouchet and Joly on the question of spontaneous generation, by Liebig on the subject of fermentation, by Germans and Italians regarding the attenuation of virus, but every one of his assertions had been met with such passionate opposition that, from sheer weariness, he had invariably ended by referring the matter to some authorised commission, only asking it to put an end to all strife by coming to some definite decision.
The upshot was at times somewhat amusing. For instance, when Pasteur described to the Academy of Medicine how, simply by lowering the temperature of a hen, he had made her susceptible to inoculation with splenic fever, the facts were at once denied by M. Colin, a professor of the school of Alfort. Pasteur immediately requested that a commission might be named, which should include both himself and his opponent among its members. This was on a Tuesday, one of the Academy days of sitting. The following Saturday, in presence of the whole commission, Pasteur produced four hens that had died of splenic fever. M. Colin himself conducted the autopsy. It was clear to everyone that their blood was full of the filaments of the splenic fever parasite. The procès-verbal was drawn up and signed by all the members of the commission, necessarily including M. Colin. The following Tuesday it was read at the sitting of the Academy. To cover his retreat M. Colin now contended that the hens had taken splenic fever not because they had been subjected to a chilling process, but because, so as to keep them in the water, the poor creatures had had their wings and feet tied to planks. This sentimental objection was disposed of by comparative experiments that had been made on hens similarly tied and inoculated, but not chilled. The latter had in no case taken the disease.
At the Academy of Sciences, some days later, a mine was sprung upon Pasteur by a posthumous publication of Claude Bernard's. He again submitted this abruptly raised question to the decision of the Academy. A series of experiments had been found among Bernard's papers, having as their object the inauguration of a new method of spontaneously generating the substance which causes the fermentation of the must of the grape.
'I will start for the Jura,' said Pasteur. 'In the midst of my vineyard, which,' he proudly added, 'is ten meters square, I will cover over some stocks with an improvised frame. These stocks will go on living and bearing grapes, which will ripen. It is now July. At this time of year, as I have already declared, the germs of the cellules which form the ferment of the grape in the vats do not yet exist, either on the green grapes, on the bunches, or on the vine leaves. I will envelop the bunches of the stocks that are underneath the frame with a layer of cotton wool that has been raised to a temperature of 150 degrees Centigrade. This done, I will come back to Paris with the keys of the frame in my pocket, not returning to the Jura until the vintage season, at the beginning of October. I predict to the Academy, that the grapes wrapped in cotton wool under the frame, and which will have grown ripe, may be crushed in the open air, and that the juice coming from them will not be capable of fermentation.'
This prediction was fulfilled. In October, Pasteur returned to the Jura, plucked off several of these stocks, laden with ripe bunches, and brought them with the utmost care to Paris. He had at last the satisfaction of depositing them intact on the table of the Academy of Sciences. He then invited M. Berthelot (editor of Bernard's pamphlet), and all his colleagues, to cut off as many bunches as they pleased. 'Only crush them in contact with pure air,' said he, 'and I defy you to produce fermentation.'
How often was Pasteur obliged to return to facts already proved, not only at the Academy of Sciences, but at the Academy of Medicine, where M. Jules Guérin, at the age of eighty, challenged him to a duel as his scientific ultimatum! If M. Pasteur at times pleaded his cause with too much passion, it was the passion of truth, the burning desire to convince, which lent such power and defiance to his vibrating voice. He could not endure his work to be attacked—not from pride, none was more modest than he—but from irritation at the denial of positive facts; facts of which he was a thousand times assured, and which all the world might verify. No one now remembers these discussions. Time has passed, and opposition has been overthrown. It has been granted to Pasteur to see, everywhere around him, the beneficent results of his discoveries. From all parts, from his own as well as from foreign countries, such proofs of admiration and gratitude have been showered upon him as are usually granted only to those whose death has atoned for their genius. He has opened up such sources of wealth to industry and agriculture that, as the learned English professor Huxley has truly said, 'Pasteur's discoveries suffice, of themselves, to cover the war indemnity of five milliards of francs paid by France to Germany.' His investigations of contagious diseases have revealed immense possibilities in prophylaxy. But Pasteur considered these marvellous discoveries as a mere beginning. 'You will see,' he often said, 'how it will all grow by-and-by. Would that my time were longer!'
THE LABORATORY OF THE ÉCOLE NORMALE.
VARIOUS STUDIES. HYDROPHOBIA.
Since the day when a minister told Pasteur, that there were not 1,500 francs in the budget to allow for the expenses of his laboratory, science has obtained a little more consideration. At the present time she has nothing to complain of: her sovereignty is recognised; her schools are becoming palaces; she has an amply sufficient civil list: she is rich enough, in short, to pay for her researches. M. Pasteur's laboratory has had its full share of the well-bestowed generosity of the State. The municipal council of Paris even wished to attach vast dependencies to this laboratory. The old garden of the ancient Collège Rollin was placed at the disposal of Pasteur; who at once hastened to build stables for lodging horses attacked by glanders, stalls for sheltering splenic fever sheep, and kennels for the reception of mad dogs. But, while taking advantage of these hospitable premises, Pasteur still retained, in the basement of his laboratory in the Rue d'Ulm, a whole population of animals under experiment. Isolated in round cages which impart some sense of security, are the rabid dogs; some attacked with furious madness, biting their bars, devouring hay, uttering doleful howls which those who have once heard can never forget; others carrying the germ of this terrible disease, still fawning with a humble look of tenderness, as if imploring attention. Hens and chickens pass their heads through the wooden bars of their coops. From time to time a cock from the bottom of his den crows 'a gloomy dawn.' Rabbits eat peaceably, while little families of guinea-pigs cluster together, and at the least alarm utter a frightened cry. All these animals are destined to be shortly inoculated. Each morning a round of inspection is made in this little hospital of condemned animals. The dead are taken out, carried to one of the upper rooms, and placed on the dissecting-boards.
It is also to such boards that living animals are fastened when it is necessary to experiment upon them. Certainly when one sees a dog lying with a forlorn look, its feet tied, its body trembling from fright, on the point of undergoing, though in full health, a bloody operation, one cannot suppress the feelings of pity. But a single visit to a physiological laboratory suffices to reveal vivisection in its only and true light; that of the interest it offers to science, and the results it may have in store for the benefit of humanity. Moreover, in Pasteur's laboratory, every dog subjected to vivisection is chloroformed. The persons who take up the controversy about vivisection are careful that the outside world shall see only the suffering and anguish of the animal, where the solution of a problem should be the object kept in view. Would the English physiologist Harvey have discovered the circulation of the blood, if he had not practised vivisection on deer in the park of Charles I.? Would Claude Bernard have been able, without vivisection, to demonstrate the glycogenic function of the liver? If Pasteur had not sacrificed some fowls and sheep, would the great scientific fact of the attenuation of virus have been discovered? If 500 dogs had to perish, what would that be, compared with the discovery tomorrow of the cause of hydrophobia, and of the means of protecting humanity against this frightful scourge?
On one occasion, in presence of a large assembly, Pasteur made an experiment on atmospheric oxygen. He placed under a glass bell a bird, which in a short time, after having consumed the oxygen contained in the bell, gathered itself up into a ball, opened its beak, and shut its eyes, as if it were going to die. At this moment Pasteur introduced a second sparrow, which, passing directly from the ordinary air into the bell, without any gradual preparation, immediately fell, asphyxiated. There was a little exclamation of horror and a movement of pity in the audience. While the first sparrow, which had gone through the ordeal unharmed, was set free, and gradually revived, Pasteur turned towards the assembly and said—
'I never had the courage to kill a bird in sport, but when it is a question of experiment I am deterred by no scruple. Science has the right to assert the sovereignty of its aims.'
But to return to the animals of the laboratory: From the little white mice, which hide themselves in a packet of wadding, to the dogs which bark furiously in their iron cages, all are devoted to death. But it is not only the inmates of the laboratory which daily succeed each other upon the operating and dissecting tables. From divers parts of France, hampers full of fowls which have died of cholera, or of some other disease, are sent to Pasteur. Here is an enormous basket packed with straw containing the dead body of a pig which had died of measles. This fragment of lung, packed in a tin box, belonged to a cow which died of peripneumonia. Other packets are still more precious. Since Pasteur went to Pauillac two years ago, to watch for the return of a ship which was to bring back some passengers attacked with yellow fever, he sometimes receives from a distant country a bottled dose of vomito negro.
Everywhere, on the work tables, are to be seen tubes filled with blood, microscope slides carrying little drops. In the stoves are ranged the cultivating flasks, which resemble little flasks of liqueur. The point of a needle dipped into one of these flasks is sufficient to cause death. Enclosed in their glass prison, millions upon millions of microbes live and multiply.
It is really a curious spectacle this workshop of research and discovery. How numerous and varied are the subjects which are being studied, and with what energy and patience does Pasteur attack them! It is not only to the most dreaded diseases that he has applied the germ theory. He has extended it to certain common disorders. Everything to him is a subject for experiment. In May 1879, a person who was working in the laboratory was troubled with boils, which reappeared, as usually happens, at short intervals, sometimes on one part of the body, sometimes on another. Pasteur, whose mind was constantly dwelling on the part played by microscopic organisms, asked himself if the pus of the boils did not contain a parasite, the presence and development of which, and its accidental transport here and there in the body, might be the cause of the local inflammation and of the formation of the pus. The constant reappearance of the evil would be thus accounted for.
The pus of the first boil, which was situated on the nape of the neck, was collected in great purity; some days afterwards, the pus of a second boil, then of a third boil, was collected. The pus, or the blood-stained lymph of the red swelling which preceded the formation of the pus, were sown in a sterilised infusion, and each time a microbe, formed of little spherical points connected in pairs, frequently united in small clusters, was seen to develop itself. The cultivating liquid was sometimes infusion of fowl, sometimes of yeast. In the infusion of yeast the little grains are suspended in pairs throughout the liquid, which is uniformly thickened with them. In the fowl infusion, the grains are united into little clusters, which cover the sides of the vessels, the liquid remaining clear as long as it is not shaken.
New observations were made upon a series of boils, in the case of a man sent to Pasteur by Dr. Maurice Raynaud. The same parasite was again found—a unique parasite, distinct from all others. At the Hospital Lariboisière, a woman whose back was covered with boils, offered another opportunity for experiment, and with the same result. It appears certain, then, that every boil contains a microscopic aerobic microbe, and that to it are due the local inflammation and the consequent formation of pus.
When guinea-pigs or rabbits are inoculated with the cultivating liquids, little abscesses are formed, which, however, quickly disappear. As long as the cure of these little abscesses is not quite completed, one can extract from them the microscopic organism which has formed them. When the little parasite is sought for in the general blood of those attacked with boils it is not found. The cause of this, no doubt, is that an aerobic parasite has always some difficulty in developing itself in the blood. The blood corpuscles appropriate, and do not willingly give up to a foreign organism, the oxygen which they require. There is a struggle for life, and in the struggle against the boils the victory is not doubtful. It might be thought, then, that the little organism of boils does not exist in the blood, but there is no doubt that if, instead of a small drop of blood, one could put several grammes or more into cultivation fruitful results would follow. The little parasite is no doubt conveyed by the blood at one time or other. It is transported from a boil, in the process of development, to another point of the body, where it may be fortuitously arrested, there to cultivate itself and form a new boil.
'It is to be wished,' said Pasteur, 'that a patient would submit to a number of punctures on different parts of the body, distant from boils already formed or in process of formation, and that with the blood thus taken from the general circulation a multitude of cultivations might be carried on. I am persuaded,' he added, 'that, among these cultivations, we should find some fruitful in the little organism of the boils.'
But whilst Dr. Maurice Raynaud gave Pasteur the means of studying boils, Dr. Lannelongue enabled him to investigate that serious disease of the bones and marrow called 'osteomyelitis.' In February 1880 that skilful surgeon, who has published a highly esteemed work on osteomyelitis, and on the possibility of its cure by trepanning the bone, followed by washings and antiseptic dressings, conducted Pasteur to the Hospice Trousseau. A little girl twelve years of age, attacked with this cruel malady, was about to be operated upon. The right knee was much swollen, as was also all the leg to below the calf, and a part of the thigh above the knee. After having chloroformed the child, Dr. Lannelongue made a long incision below the knee, from which pus flowed abundantly. The bone of the tibia was laid bare for a considerable length. Three trepanning perforations were then made in the bone, from each of which the pus issued in great quantities. Pasteur carefully collected, with all the conditions necessary to the preservation of their purity, the pus of the exterior and the pus of the interior of the bone, and, returning to his laboratory, he examined them attentively. The direct observation, by a microscope, of the two specimens of pus was extremely interesting. It was obvious that they contained, in large quantities, an organism like that of boils, in pairs of two or four, and also in parcels, some with a clearly defined outline, others scarcely visible, and with very faint outlines. The external pus showed an abundance of pus globules, but that of the interior did not show any. It was like a paste entirely composed of microbes, so numerous and fertile that, in less than six hours after sowing them in the cultivating liquid, the development of the little microbe had commenced, and was rendered visible to the naked eye by a slight but general turbidity of the liquid.
Its close resemblance to the organism of the boil might lead to the assertion that they are identical, if it were not known how great are the physiological differences that may exist between microscopic parasites of the same appearance and the same dimensions.