On the Anthrax Inoculation

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I am indebted to Professor K. Codell Carter of Brigham Young University for allowing me to use his translation of the following essay, taken from his book "Essays of Robert Koch", Greenwood Press, N.Y., 1987.

On the Anthrax Inoculation [1872]
by Robert Koch
(Ueber die Milzbrandimfung, Leipzig, Georg Thieme, 1882)

    In one of the plenary sessions of the Fourth International Congress for Hygiene that was held this September in Geneva, Pasteur delivered a lecture on the attenuation of inoculation material.1 As a member of the Congress, I certainly did not miss the opportunity to attend this lecture. I was anxious to hear valuable scientific results from Pasteur's work in attenuating anthrax bacilli. I wanted reliable figures about losses after preventive inoculations and about the resistance of inoculated animals to natural infection. I also hoped that important new discoveries about infectious diseases would be communicated. I knew that during the preceding year, Pasteur had been occupied with the occurrence of yellow ever in southern France, and that he had sought the microbes of Lungenaeuche.

    But the Congress heard none of this. One heard only already familiar things about chicken cholera, about the nouvelle maladie de la rage and, with respect to preventive inoculations for anthrax, only the worthless fact that as yet so-and-so many thousand animals had been inoculated. Only one thing appeared to be new: a parasite that Pasteur supposedly discovered in typhoid fever of horses but which, as we will see, is already known. All of this provided only an opportunity for a polemic against me; a polemic that was not limited to the announce theme, but that concerned all the differences in our opinions of the etiology of anthrax. Such matters as the cooling of chickens and the significance of earthworms have, in my opinion, already been settled, but in any case they have no significance for hygiene, and they certainly should not be discussed in a plenary session of a congress on hygiene. This is particularly the case since Pasteur's polemic did not involve a factual refutation, but consisted of generalities. It was mostly directed against me personally, and was delivered in an emotional tone. It seemed most prudent, therefore, to restrict myself to a short protest against Pasteur's attack, and to postpone a detailed response.2  In presenting this response, it is appropriate to explain my position with respect to Pasteur's work on the infectious diseases. It is unnecessary to point out that even though I must contradict Pasteur on many specific points, I certainly do not deny that in other areas he has provided meaningful service in the interest of science.

    There are fundamental differences between the methods that Pasteur and I use in studying infectious diseases. For this reason alone, it was inevitable that our experimental investigations would lead to divergent results.

    My point of view can be characterized briefly as follows: It is not yet proven that all infectious diseases are caused by microorganisms, and therefore, one must demonstrate the parasitic nature of each particular disease. The first step in this demonstration is the conscientious examination of all those body parts that are altered by the disease. In this way, one can establish the presence of the parasites, their distribution in the diseased organs, and their relation to the body tissues. Of course, in this investigation one employs all the resources of modern microscopic technology. One must microscopically examine fresh tissues and such bode fluids as blood and lymphatic fluid both with and without the use of reagents; one then dries them under glass and treats them with various stains. The hardened matter is also finely sliced by a microtome, and stained. One thoroughly examines the resulting microscopic preparations with appropriate lighting and with the best lens systems. In this way one achieves a complete orientation regarding whether microorganisms are in the diseased organs and in which organs. Only then can one begin to demonstrate that the microorganisms are pathogenic and that they cause the disease. To do this they must be cultured in pure form. Then, after they have been freed from all the parts of the diseased body with which they were originally associated, they must be inoculated back into animals of the same species as those in which the disease was originally observed. At least they must be inoculated into animals that are known to display the symptoms of the disease in question. As an example consider tuberculosis.3 First, by microscopic examination, bacilli were demonstrated in diseased organs. These bacilli could be precisely characterized by their reaction to stains. They were isolated in pure cultures where they could not be contaminated by other bacteria. Finally, the pure culture was reinoculated into as many different kinds of susceptible animals as possible. A second. very instructive example is human erysipelas. For a long time it was known that in this disease the lymphatic veins of the skin contained specific micrococci. However, this did not prove that these micrococci were the cause of erysipelas. Using every possible precaution against contamination by other bacteria, [Friedrich A.] Fehleisen recently obtained a pure culture of this micrococcus from pieces of skin excised from victims.4 He then generated typical erysipelas infection by inoculating humans. After this, no question remains that the micrococcus is the cause of erysipelas, and that the disease is parasitic.

    Pasteur departs significantly from this approach, which, from my point of view, is the only one that satisfies current scientific standards.

    First, Pasteur assumes that all infectious diseases are parasitic and caused by microbes. Apparently, he regards it as unnecessary to establish the presence and distribution of microorganisms in the body--the first of the conditions that I have mentioned. Thus, Pasteur made no claims about discovering a specific microbe in the organs, particularly in the sublingual glands, of the child who died of what Pasteur called the nouvelle maladie de la rage. Yet these glands provided the point of departure for his infection experiments. Precisely in this case, such an investigation is absolutely essential because it is known that the sublingual glands contain infectious material for rabies. Moreover, since these tissues do not ordinarily contain bacteria, they would be the most likely place to discover the supposed microbes in their purest form. But in his attempt to transmit rabies from the corpse of the child, Pasteur used saliva rather than the tissues of these glands. Saliva is known to contain many different bacteria. [Edme-Felix-Albert] Vulpian and (George Miller] Sternberg have shown that even saliva from healthy persons contains pathogenic bacteria.5  Pasteur approached so-called horse typhoid fever in the same way. He did not examine the edematous swelling of the skin and intestine or the swollen spleen. We were not told whether the blood of ill or dead animals contained characteristic microbes. Pasteur was content to inoculate with slime taken from the nose of the dead animal, which, exactly like saliva, was certainly contaminated with many other bacteria.

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    Pasteur began with impure material, and it is questionable whether inoculations with such material could cause the disease in question. But Pasteur made the results of his experiment even more dubious by inoculating, instead of an animal known to be susceptible to the disease, the first species that came along--the rabbit. To determine whether some substance contains rabies poison, one must first inoculate dogs. Suppose one is exploring the etiology of some new horse disease. Even if one inoculates with nose slime, which is certain to be contaminated by other bacteria, rather than with blood or spleen tissue, one must at least use horses as test animals. No one knows whether rabbits can even contract horse typhoid fever or what symptoms they would have if they did.

    The consequences of Pasteur's method were striking. Pasteur found that anthrax was already understood. The anthrax bacilli were known, and it had been established that they caused the disease. The weaknesses of his method were less apparent in this case than in his later work because the facts were already known. In his work on chicken cholera, the way had been prepared by [Edoardo] Perroncito and [H.] Toussaint;6 once again he could not go far astray. However, as soon as Pasteur turned to the completely new subject of rabies, he was immediately misled by his defective method. Pasteur did not discover rabies microbes; apparently, they are still sought in vain. Instead, he found bacteria that supposedly caused a new disease. However, if one carefully examines this new disease, it turns out to be rabbit septicemia. Pasteur described the new microbes as small, somewhat elongated, and slightly tapered in the middle so that they resemble the numeral eight. Rabbits infected with the microbe died in twenty-four hours. By appearance and by pathological operation it is apparent that these microbes are precisely those of rabbit septicemia. [Leon] Coze and [Victor Timoth6e] Feltz and later [Casimir] Davaine and finally [Georg] Gaffky have all made thorough experimental studies of this microbe.7  It is remarkable that the pathological process that we call septicemia is not a single disease. This has been established ever more conclusively in studies of pathogenic bacteria. There are various different pathogenic bacteria that attack specific animal species and cause fatal diseases with the symptoms of septicemia. We are already familiar with a small bacillus that causes septicemia in mice. This bacillus never kills guinea pigs, and in rabbits it causes a process like erysipelas. I also repeatedly observed a very small micrococcus that caused characteristic septicemia of guinea pigs. Moreover, the previously mentioned 8-shaped microbe that caused rabbit septicemia was observed after inoculating rabbits with various decaying substances. Until now, it has been obtained by inoculations with decaying blood, gutter water, and other decaying liquids. Sternberg regularly caused this disease with his own saliva,8 although he was in good health. He published characteristic photographs of the 8-shaped microbe which he obtained by inoculations with his own saliva. Every experimenter who injects rabbits must be aware of these facts if he wishes to avoid error. Either Pasteur did not know these things or he ignored them. Otherwise, he would not have injected rabbits with a fluid as rich in microbes as is the saliva of corpses. Instead, he would have injected dogs; they are only weakly susceptible to septicemia infections. In any case, he would not have identified as a novelle maladie a disease that has long been known. It is inconceivable that Pasteur, having failed to learn from this experience, would fall into the same errors in his investigation of horse typhoid. It is of no relevance to the etiology of this disease that a dog dies shortly after being inoculated with the nose slime of a dead horse. Just as rabbits can be killed with saliva from healthy humans, one could surmise that decaying fluids from a horse that died of some disease or other would also be fatal. One should first have determined whether inoculations with the nose slime of other horses, especially healthy ones, would kill rabbits. All this was neglected. By a droll coincidence, the fateful 8-shaped microbe, which kills rabbits in approx- imately twenty-four hours, appeared. Those who are familiar with the infectious diseases of animals will immediately recognize that rabbit septicemia, which Davaine has described, and the disease that Pasteur call. a novelle maladie are identical. Actually, it could not be otherwise because rabbits react with septicemia if they are injected with fluids containing this specific bacterium. If Pasteur procedes in this way, by injecting dogs with decomposing animal fluids, I have no doubt that he will again encounter the 8-shaped microbe of rabbit septicemia. He will have further opportunities to present this to Academies and Congresses as highly interesting and eminently important. But, even if the inoculation of horse-nose mucous were to produce a variant form of a previously well-known artificial infectious rabbit disease, I would still regard this as of minor importance It would not be a suitable matter to communicate to a world congress. In the course of etiological investigations conducted in the Imperial Gesundheitsamt, approximately ten different artificial infectious animal disease have been discovered. These were not caused by 8-shaped microbes, but by characteristically formed species of bacteria that determine unique pathological processes. However, the pathological microorganisms of naturallly occurring infectious diseases seem to us to be much more interesting, and we do not find it appropriate to make each of these discoveries the subject of a detailed public announcement.

    Thus, because of the lack of microscopic investigation, because of the use of impure substances, and because he used unsuitable test animals. Pasteur's method must be rejected as defective. It cannot lead to conclusive results. One cannot criticize Pasteur himself for his interpretation of his results. His biases got the better of him, and he reported wonderful things about the diseases found in his test animals and about the remains in their corpses. After all, Pasteur is not a physician, and one cannot expect him to make sound judgments about pathological processes and the symptoms of diseases. By so much the more, therefore, were his medical associates obliged to warn him against the grossest errors. They might have warned him about his conception of the rabbit disease that came from injections of nose slime from horses. According to [Wilhelm] Schütz's investigation, this horse disease is an erysipelatous process and has nothing to do with human typhoid.9  Also, as already mentioned, the disease caused by inoculation with nose slime is identical to rabbit septicemia and is also unrelated to typhoid. This is proved by the microbes en huit and by the rapidly fatal influence of the parasites. However, the accidental choice of the name 'horse typhoid' seems to have led Pasteur to imagine that this was a genuine typhoid disease, possibly even a disease closely related to or identical with abdominal typhoid. He especially emphasized that infected rabbits had swollen Peyer glands, particularly in the region of the ileocecal valve, and that the animals died in less than twenty-four hours of a veritable fièvre typhoide!. There is no indication of how this fever was diagnosed as typhoid. But even without further explanation, the whole matter takes on a wonderous appearance because as yet neither rabbit typhoid fever nor typhoid fever lasting only one day has ever been encountered. This one-day rabbit typhoid belongs with the one-day rabbit rabies of [Obidou] Lannelongue and [Maurice] Raynaud and with the swine syphilis briefly described by [Louis] Martineau and [Paul] Hamonic in which, one day after inoculation, bacilli were in the blood.10  These things directly contradict all experience and the current opinions of science; they can only erode the confidence that etiological investigation is beginning to enjoy. It would be better for the development of this young branch of science if such errors could either be quickly refuted or totally forgotten. One can only wonder that a periodical so well edited as the Annales d'hygiène publique can have seriously reported that Pasteur had cultivated typhoid bacteria.11 In this context, every reader must conclude that the claim concerns the bacteria of true abdominal typhoid.

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    In his Geneva lecture, Pasteur bitterly complained about my having rejected his microscopic examinations and inoculation techniques. However, after his inoculations with saliva and nose slime and his repeated discovery of the microbe en huit, I am not able to change my opinion.

    Pasteur deserves criticism not only for his defective methods, but also for the way in which he has publicized his investigations. In industry it may be permissible or even necessary to keep secret the procedures that lead to a discovery. However, in science different customs prevail. Anyone who expects to be accepted in the scientific community must publish his methods, so that everyone is able to test the accuracy of his claims. Pasteur has not met this obligation. Even in his publications on chicken cholera, he attempted to keep secret his method of reducing virulence. Only pressure from [Gabriel Constant] Colin induced him to reveal his methods. The same was true of his attenuation of the anthrax virus. As yet, Pasteur's publications on the preparation of the two inoculation materials are so imperfect that it is impossible to repeat his experiments and to test his results. Having adopted such a procedure, Pasteur cannot complain if he encounters mistrust and sharp criticism in scientific circles. Science makes its methods public without reservation. In this respect, Toussaint and [Augusta] Chauveau, who are working in the same area, are a pleasing contrast to Pasteur.

    After this explanation, which shows that there are reasons enough to criticize Pasteur, I turn to a discussion of the chief disagreements between Pasteur and myself. These concern the etiology of anthrax, and questions about the attenuation of anthrax virus and about artificial immunity to anthrax.

    I can quickly dispense with the etiology of anthrax because Pasteur has not answered even one of the objections I raised against his conception. He responded with general phrases instead of facts and, of course, such language does not change matters at all. In science, facts are decisive; beautiful and well-constructed speech is not.

    Pasteur believes that he has discovered the etiology of anthrax. This etiology could only be established by identifying the enduring forms of anthrax bacilli, the conditions of their origin, their characteristics, and their relation to soil and water. Although I have no interest in priority disputes, these matters are so obvious that I cannot ignore them. I can only answer Pasteur's claims by referring to my publication of 1876 that describes the generation of anthrax spores and their relation to the etiology of anthrax. Pasteur's first work on anthrax was published one year later, in 1877. This requires no further comment.

    Pasteur claims that birds are immune to anthrax because of their higher blood temperatures, but that if they are permanently cooled a few degrees, they become susceptible. To prove this, he nailed chickens to a board and placed them in cold water. Against this experiment, I objected that chickens are not totally immune. [H.] Oemler's experiments prove that chickens can contract anthrax, and that other birds, such as sparrows, in spite of their higher blood temperature, can also be infected with it.12. Pasteur objected that I should at least have repeated his experiment before challenging it. Evidently he misunderstood my criticism. I do not question the factual aspects of his experiment, but rather its significance. I remain by my opinion because, as I said, other birds contract anthrax in spite of their higher blood temperature. I have confirmed this by several of my own experiments. Moreover, nailing chickens to a board and immersing them in water is such an extreme violation of their living conditions, that cooling alone may not have made them more susceptible to anthrax; susceptibility may have been partly due to disruptive trauma. In preventive anthrax inoculations, weak animals generally die. Thus, reducing the life forces of an animal can make it more vulnerable to anthrax. Something similar may have happened to the nailed-up chickens. In any case, the experiment was not pure. It did not prove what Pasteur intended to prove, and I feel no need to repeat this useless experiment.

    A much more important difference of opinion concerns the occurrence of natural infection. Pasteur assumes that spores form in buried anthrax cadavers, that they are brought to the surface by earthworms, and that they are deposited on fodder with dust. According to Pasteur, fodder must be prickley to cause an infection. It does this by wounding the mouth of the animal, and the infection proceeds as a kind of inoculation in the mouth cavity. Pasteur hopes to prove this by observing that glands in the lower jaw of animals that die of spontaneous anthrax are regularly swollen. This is supposedly because they are nearest to the infection site. However, according to my observations, anthrax bacilli can multiply and form spores on decaying vegetable matter independently of animal cadavers. Apparently, they live in this way in swampy areas. Experience shows that animals in such areas often become infected even where anthrax cadavers have never been buried. Thus, the danger of infection is not restricted to anthrax cadavers, and earthworms are superfluous in the spread of anthrax. Other considerations, for example, low ground temperatures in many lands, such as Siberia, in which anthrax is most prevalent, also count against Pasteur's theory. I have also conducted direct experiments with earthworms, and my results do not confirm Pasteur's assumptions. Moreover, I must contest the claim that natural infection presupposes prickley fodder and small injuries in the mouth. I have conducted experiments that show this to be false.

     I turn now to the attenuation of the anthrax virus and the artificial immunity that it supposedly confers.

    Pasteur first undertook experiments with the attenuation of the chicken cholera microbe. This convinced him that attenuation was due to oxygen. He then applied his experience to the anthrax bacillus, and he succeeded in weakening it as well. Test animals withstood inoculation, and after inoculation they were immune to inoculation with the strongest anthrax poison.

    Pasteur found that two preventive inoculations were necessary to immunize animals against unweakened anthrax. One first inoculates highly weakened vaccine, then one uses a vaccine that is somewhat stronger.

    His first success with chicken cholera gave Pasteur the most farreaching hopes. Once he had made a few sheep immune to anthrax by preventive inoculations, he did not hesitate to ascribe a universal significance to his experiment. To him it seemed certain that all animals could be made immune in this way, and that all other infectious diseases would be exactly like anthrax. Their associated microbes could be attenuated and converted into protective vaccine. With complete confidence, he announced imminent victory over the infectious diseases. (Friedrich] Löffler's work on immunity was conducted at this time in the laboratories of the Gesundheitsamt.13  Pasteur communicated his results so imperfectly, that it required the most comprehensive study to repeat and test his work. Thus, Löffler focused mainly on immunity in general. After numerous experiments with mice, rabbits, rats, and guinea pigs, he concluded that there are, in fact, bacterial diseases to which an individual can become immune by withstanding the disease. However, several bacterial diseases can attack the same individual, more than once. Thus, these diseases do not provide protection against subsequent infection. Löffler asserted that the animal specles with which he experimented did not become immune to anthrax. However, he did not conduct experiments with sheep; and he felt that further tests with these animals were necessary. Only in this way could one determine whether the hope awakened by Pasteur's experiments in Pouilly-le-Fort would be fulfilled. This opinion, which conformed completely to what was then known, has subsequently been verified.  Löffler's statements about anthrax have been completely confirmed.

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    From chicken cholera and anthrax, Pasteur generalized conclusions about all infectious diseases. These conclusions cannot be harmonized with the experience of medical science. In addition to the results of his experiments with artificial infectious animal diseases, Löffler also mentioned erysipelas, gonorrhea, and relapsing fever. Each of these has been proved to be an infectious disease caused by bacteria, but experience shows that victims of these diseases are not immune to new infections. Tuberculosis has recently been added to the diseases that can befall humans more than once. No physician has claimed that a tuberculous person suffering from scrofulous or fungal disorders of the limbs, for example, would, if healed, be protected against tuberculosis. On the contrary, experience shows that such persons have an increased susceptibility for tuberculosis, and later they frequently become phthisic. As yet, there are no experiences to suggest that one can become immune to leprosy, which is unquestionably a bacterial disease. Thus, experience shows that what Pasteur has assumed to be a universal law, does not, in fact, have this status.

    Even in respect to anthrax, the law of immunity does not have the breadth of application that Pasteur assumes. Löffler found that guinea pigs, rats, rabbits, and mice cannot be made immune, and this point has been confirmed by all the researchers who have given it their attention. [Alfredo] Gotti in Bologna conducted protective inoculations on several animals including six rabbits.14 When they were inoculated with anthrax blood, all the rabbits died of anthrax. [Alfred] Guillebeau also found that rabbits which had been inoculated with Pasteur's preventive inoculation matter died of anthrax after inoculations with anthrax blood.15  Klein undertook tests on guinea pigs and mice with inoculation matter that he obtained in Paris; all the animals died of anthrax.16 In the
Gesundheitsamt we conducted numerous experiments on rabbits, guinea pigs, and mice with virus that had been attenuated to various degrees. We also used Pasteur's genuine inoculation matter. In spite of all our efforts, we never succeeded in making these animals immune to unweakened anthrax poison. They all died of anthrax after the control inoculation. Thus, not all animal species can be immunized by Pasteur's procedure. Apparently, horses cannot be protected in this way. Several unsuccessful attempts to inoculate horses were discussed at the meeting of the Société centrale de medecine vétérinaire on 8 June 1882.17  There are also other reports that horses do not endure preventive inoculations. Löffler gave a series of examples showing that humans apparently do not become immune to anthrax by withstanding the disease.18 More recently, J. de Jarnowsky published further evidence; in his own practice he observed fifty anthrax victims.19 He mentioned one person who had the disease twice in the course of two years, and another who had it three times in three years.

    Until now, preventive inoculations have achieved a pronounced immunity only among sheep and cattle. For the time being, these are the only species that benefit from such inoculations. According to Pasteur, his experience shows preventive inoculations of sheep and cattle are safe and reliable and provide enduring protection. In fact, Pasteur's experiments have been given numerous practical tests. It remains to be seen whether Pasteur's promises about the safety and reliability of the inoculation will be fulfilled. These matters are central to the immunity question; they require thorough discussion.

    Many reliable observers have tested Pasteur's inoculation matter in different places. All their work is relevant to appraising the effectiveness of the procedure. However, these experiments were entirely practical in orientation, and several important circumstances have been ignored. Consequently, I must give special weight to investigations of anthrax immunity conducted this year at the Imperial Gesundheitsamt. Dr. Löffler, Dr. Gaffky and I conducted these experiments together. In this context I can only summarize our results, but we intend soon to publish a more complete account.20 We were not able to use such an impressive series of test animals as Pasteur's financial support enabled him to use. In spite of the relatively limited number of our test animals, we hoped to resolve a few important questions about the etiology of anthrax and about artificial immunity.

    With respect to the preparation of the inoculation matter, Pasteur said only that anthrax bacilli were cultivated in a neutralized meat broth at 42o to 43oC.  After twenty days, the bacilli were sufficiently attenuated that they could be used for the first inoculation of sheep. Pasteur said nothing definite about the best time to obtain the second vaccine, or about how to judge its degree of attenuation. Yet these are crucial matters. Many people would like to know more about creating vaccine, and so I will describe our experiences in this area. It is essential to have a thermostat that can maintain a constant temperature without the slightest variation over a period of weeks. Following d'Arsonval, we used an instrument constructed in Paris by Wiesnegg. With the usual precautions, we placed into the instrument a small Mask that held about twenty grams of liquid. The flask contained chicken broth that had been infected with anthrax bacilli, and it was maintained at 42.5oC. Every second day we took material from the flask to inoculate mice, adult guinea pigs, and large strong rabbits; at the same time, we created a pure culture in gelatin from the same liquid. Originally, all the animals died of anthrax after the inoculation. After several days--the number of days was not constant in all the tests and often differed from container to container in the same test--the inoculation of the rabbits became uncertain. Only some of the inoculated rabbits died. For example, if three or four were inoculated, one or two would die. All the mice and guinea pigs were killed by the inoculation. Still later, guinea pigs withstood the inoculation while mice continued to die. Finally, we obtained a pure culture of anthrax bacilli from which even mice could be inoculated without harm. These harmless anthrax bacilli were morphologically the same as virulent bacilli. They were completely nonmotile, and in pure culture built long filaments exactly like virulent bacilli. Cultures that were fatal to mice but not to guinea pigs provided the best material for the first inoculation of sheep. The material for the second inoculation could be taken from cultures that killed guinea pigs but did not consistently kill rabbits. Of course, there are other degrees of virulence that can be useful for vaccines if one is not limited to two inoculations. I doubt that Pasteur knew how to recognize these levels of virulence; if he had, the strength of his vaccine would not have been so variable. I tested one of Pasteur's first vaccines that would not kill mice; thus, it was too weak. I tested one of his second vaccines that killed all the large rabbits that were inoculated with it; it was too strong. Klein used first inoculation material that he obtained from Boutrous, Pasteur's agent." He inoculated four guinea pigs and four mice. Within forty-eight hours, three guinea pigs and all the mice died. This showed that the inoculation matter was too strong for the first vaccine. According to a report in the Wiener landwirtschaftlichen Zeitung, twenty-two sheep were inoculated in Hungary with the second vaccine without having been inoculated with the first.22 Yet, all the animals remained healthy. One can surmise that this inoculation material was too weak.

    The temperature at which the culture is maintained has a great influence on the time required for attenuation. The nearer the temperature is to 43o°C, the more quickly attenuation occurs. It can be completed in six days. At 42oC the process can require thirty days. For this reason, it is essential to test the vaccine on mice, guinea pigs, and rabbits. If the culture is maintained for a long time at room temperature, it gradually looses its virulence. Pasteur made this observation, and we have confirmed it several times. We transplanted attenuated cultures into gelatin media. From these cultures we created large quantities of pure cultured inoculation fluids. The purity of the cultures is vital because the invasion of foreign bacteria, among which some may be pathogenic, seriously increases the danger of inoculation. Some of the failures of preventive inoculations of horses may have been due to contamination of the inoculation material by septic bacteria. In microscopic examinations, I found Pasteur's original vaccine to be contaminated with numerous kinds of bacteria.

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    Both the vaccine that we made ourselves in this way, and the vaccine that we obtained in Paris from Pasteur's agent were used exactly according to Pasteur's instructions. We carried out inoculation tests on sheep; these had the following results: There was almost no reaction to the first vaccine, which killed mice but not guinea pigs. After the second vaccine, a few animals died of anthrax. Because of the small number of test animals, there is no point in giving percentages. In general, our results agreed with the results of tests conducted in Kapuvar and Packisch.23  I will occasionally compare these tests with ours, since both series of tests were reliably observed and controlled by a commission named for that purpose. In Kapuvar, of fifty sheep, none died after inoculation with the first vaccine; after the second vaccine, five sheep died.  In Packisch, the first inoculation caused no losses, but three of twenty-five sheep died of anthrax after the second. Similar figures have been reported in numerous other inoculation tests. From the facts, one can assume that there will be no losses after the first inoculation, but ten to fifteen percent after the second. Pasteur regarded these losses as unusually high, and attributed them to the use of sheep that were particularly susceptible. However, recent reports from France indicate large losses.24 Thus, there seems to be no significant difference in resistance between our sheep and French sheep. Preventive inoculations involving several thousand animals have been conducted in France, and these resulted in very few losses. But the immunity of these animals was not tested by impeccable control inoculations. They may have been injected with a weaker vaccine and, therefore, have had reduced protection. As Pasteur explicitly reported, in the second test in Packisch, in which 251 sheep were inoculated, weaker inoculation matter had been provided and, consequently, only one sheep died of anthrax.

    Of course, after our preventive inoculations, we conducted control inoculations with unweakened anthrax poison. In one case, three weeks after the last inoculation, of six sheep that had been inoculated with Pasteur's vaccine, strictly according to his instructions, one died of anthrax. Two sheep inoculated with a different preventive vaccine remained alive after the same inoculation. These numbers are also too small to speak of specific proportions. But it is significant that in Packisch, twenty-two control sheep all remained healthy and in Kapuval one of forty-four sheep contracted anthrax, while we lost one sheep in six. The explanation may be that the control tests in Packisch and Kapuvar employed virulent anthrax matter that Pasteur had prepared in Paris specifically for that purpose, while in our control inoculation we used anthrax poison prepared locally. Our material may have been more virulent than Pasteur's.

    In addition to our own tests, the following facts support this opinion.

   Saake, the district veterinarian in Wolfenbüttel, reported that eighty-two sheep in Salzdahlum were inoculated with Pasteur's first and second vaccines.25  Three sheep died after the second inoculation. Thus, the vaccine was sufficiently strong. Eight weeks later, ten of these sheep were given a control inoculation; two died of genuine anthrax. In this test, control inoculations were made with anthrax blood taken from a sheep that happened to die of spontaneous anthrax.

    Tests conducted by Bassi in Turin revealed a clear difference in virulence between the so-called unweakened anthrax poison that Pasteur provided for control, and the poison taken from animals that died of spontaneous anthrax.2g Six sheep that had been given preventive inoculations were injected with Pasteur's virulent matter; all remained healthy. Six others that had also been given preventive inoculations were injected with blood from a cow that had died of anthrax two-and-one-half hours earlier; two died of anthrax.

    I explain these striking occurrences by the possibility that Pasteur's unweakened virus gradually became weakened, exactly like his vaccine, and in time no longer had its original strength.

    Thus, tests in Salzdahlum and in Turin, as well as our own, show that a significant number of sheep withstood the second vaccine but died after inoculation with local anthrax poison. Thus, they were not completely immune. One must surmise that inoculation with an attenuated second vaccine, which kills only reduced numbers of sheep, would also provide diminished protection. In fact, it becomes ever clearer that this suspicion is correct. According to Pasteur, in France by the beginning of September, 400,000 sheep and 40,000 cattle had been inoculated. Pasteur estimated losses to be about three sheep per thousand and about 0.5 cattle per thousand. Of course, I will not challenge these figures, but it is necessary for them to be accompanied by a commentary. From these figures, one knows only that a relatively large number of animals withstood inoculation. However, Pasteur says nothing about our main concern, namely whether the inoculations fulfilled their purpose and made the animals immune. The value of preventive inoculations is determined by how many animals have been immunized. What would one have said of Jenner if he could claim no advantage for inoculation except that of thousands of inoculated children, only such-and-such a percentage died? Certainly, nothing would do more to insure acceptance of anthrax inoculations than knowing that thousands of animals have been protected against anthrax. As yet, Pasteur has not been able to show this. On the contrary, complaints about failures of the inoculation are accumulating, and its weaknesses are becoming ever more obvious.

    On 8 June, in a meeting of the Société centrale de médecine uétérinaire, a number of failures were discussed, and Pasteur was questioned about them.27 He explained that these and many others were known to him. The reason for the failures was that the originally cultivated inoculation matter had gradually lost its virulence, and that through the winter until the end of last March he provided vaccine that had become too weak. We learn from this that during a long period of time, inoculations were conducted with matter that was too weak. We can no longer be surprised that so few sheep were lost of the hundreds of thousands that were inoculated in France last winter. On the other hand, it is very surprising that Pasteur, who conscientiously included the animas inoculated with weak vaccine in order to arrive at the largest number of inoculations with the smallest number of losses, completely ignored the many failures that were known to him. Pasteur's explanation of the failures was also wrong. If Pasteur were correct, the inoculations from the beginning of this April would have had a more uniform effect, and reduced mortality would show that more adequate protection was being provided. The following notes regarding some of the inoculations undertaken after the beginning of April, however, show that this has not been the case.

    In Turin, Bassi administered the first inoculation on 20 April, and the second on 5 May. After control inoculations with unweakened anthrax, two of six sheep died 28.

    The inoculations in Salzdahlum, which have already been mentioned, occurred between 25 May and 9 August of this year. There were two fatalities from ten control inoculations, and four percent were lost at the second inoculation.

    In June of this year, twenty-two sheep were inoculated in Hungary with the second vaccine which was apparently too weak. This case was also discussed above.

    The Recueil de médecine vétérinaire reported that between 25 April and 8 May of this year, 296 mutton were inoculated.29 Of these, only one animal died of anthrax and that was ten days after the second inoculation. Evidently, the inoculation matter was too weak. Four animals died between 22 and 24 June of spontaneous anthrax. This was all the more striking given that there were no losses among eight mutton that were not inoculated, and that were used as controls.

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    In Montpothier the inoculations took the following wondrous course. On 18 April of this year, 220 mutton were given the first vaccine; nine animals died. On 29 April the same animals were given the first vaccine once again; seven died. On 17 May, they were inoculated with the second vaccine; one died. One would have surmised that after these three inoculations with significant losses, the herd would be protected against anthrax. However, this proved to be false. Between 11 and 13 June, six mutton died spontaneously of anthrax. It was decided that the second inoculation should be repeated. This was done on 17 June; five more mutton died. One could ask whether artificial immunity is at all possible, or whether one is simply deceived by the inoculation matter.

    The inoculation in Packisch also came in this period. The inoculation matter in the first series was clearly too strong because twelve percent died. That of the second series, which Pasteur identified as weaker, was too weak because, as I will show later, it did not protect against natural infection.

    These examples show that even after the beginning of April, Pasteur's matter was sometimes too weak and sometimes too strong. It was even less reliable than the material he provided last winter.

    In the meeting of the Societé centrale de médecine vétérinaire on 8 June, Pasteur clearly felt the precariousness of his position. If he provided strong inoculation matter, which alone protected against inoculated anthrax (at least against inoculation with Pasteur's so-called virulent matter), then too many animals died after the second inoculation. However, if he provided weak material, as he did last winter, then it evidently failed to provide sufficient protection. To free himself from this difficulty, Pasteur advanced the noteworthy claim that it was not necessary to treat sheep with inoculation matter that occasioned so many fatalities because inoculated anthrax was more dangerous than natural infection. A weak vaccine was sufficient to protect against natural infection. Pasteur did not tell us what grounds he had for this claim, which was obviously arbitrary and intended only to save the endangered program of protective inoculations. Actually, Pasteur should first have decided whether inoculation really protected animals from natural infection. This should have been done before inoculations were begun, and not after hundreds of thousands of animals were inoculated with significant losses. Suppose, in contrast to Pasteur's assumption, inoculated animals were at least partially protected against artificial infections but not against natural infection. What benefit would the whole program then have? Since this question was certainly crucial for calculating the value of artificial anthrax immunity, it would have been resolved before any experiments were conducted in the Gesundheitsamt. We would not have been concerned only about making a large number of sheep immune to inoculated anthrax. The reality of artificial immunity could no longer be doubted after animals had been made immune by Toussaint and Pasteur. Before anything else, we would have determined how natural anthrax occurred.

    As mentioned before, Pasteur assumed that infection was caused by rough prickley fodder that injured the mouths of animals. Ultimately, this was a particular kind of inoculation anthrax. On an earlier occasion, I mentioned various considerations that counted against this conception. It is not necessary to repeat these considerations here because I will now depict various experiments that directly contradict Pasteur's theory.

    I fed several sheep fodder that contained anthrax bacilli but no spores. A few other sheep were fed anthrax masses that contained spores. I fed the sheep by carefully placing pieces of potato into their mouths. These pieces were filled with infection matter. They were inserted so that there was no possibility of injuring the mucous membrane. Certainly a piece of potato cannot count as prickley food. Otherwise, the sheep were fed only soft hay. Thus, Pasteur's conditions for infection were totally excluded. As a spore-free substance, we used the fresh spleen of a guinea pig that had just died of anthrax. As a substance containing spores, we used a culture of anthrax bacilli on potato that was building spores. The sheep fed with spore-free spleens remained healthy. Within a few days, the sheep fed with bacilli cultures that contained spores all died of anthrax. Dissections of these sheep showed unmistakably that the in(bction had proceded from the intestine. In the mouth cavity, in the pharynx, and in the esophagus, not the slightest injury or alteration was found that would indicate that infection had occurred there. Apparently, anthrax bacilli are destroyed by stomach acids, while spores pass through unharmed. They grow in the alkaline content of the intestine and then penetrate the mucous membrane of the intestine. Microscopic investigation suggests that lymph follicles and Peyer's glands constitute the invasion sites.

    I fed these sheep anthrax cultures that contained large collections of fresh spores. Subsequently, sheep were also fed masses containing spores that had been preserved dry for more than a year. These sheep died of anthrax as reliably as those fed with masses containing fresh spores or inoculated with fresh anthrax blood. To establish the possibility of infection from the intestine, we used a fairly large quantity of spore material in these tests. It was, therefore, necessary to determine that natural infection could also occur from eating a small quantity of anthrax spores mixed with fodder as dust or mud from a swamp or flooding stream. For this reason, we undertook the following experiment: Each day, ten sheep were given pieces of potato to which silk threads with anthrax spores were attached. Each thread was scarcely one centimeter long. One year earlier, each thread had been impregnate with a small quantity of anthrax spores and then dried. Two sheep, which served as controls, were kept in the same stall with the other sheep, and were cared for in the same way. However, they received no threads containing spores. Four of the ten sheep died. These deaths were on the fifth, sixth, eleventh, and nineteenth days of the test. The feeding tests were then discontinued. Both control animals remained healthy. In this test, the cases of anthrax that occurred over several days and the dissection results conformed perfectly to natural anthrax. Natural infection usually--in cold weather perhaps always--occurs when fodder containing anthrax spores enters the intestine. If sheep eat fodder that contains many spores, they die after a few days; if they eat fewer spores, they die more slowly. This can be explained by assuming that the ingested spores do not all develop in the intestine, but that most of them pass through unchanged. Excretement from sheep that consumed sores contained many ungerminated spores. I established this by inoculations with dried excretement that was one year old. Thus, if only part of the spores germinate in the intestine, the more spores that are ingested, the more quickly and reliably will infection occur. The number of spores that germinate will vary directly with the number of spores introduced into the intestinal canal.

    Dissections of animals that die of intestinal anthrax, as well as dissections of sheep killed by inoculated anthrax, reveal a noteworthy fact. The swelling of the lymph glands is variable and only rarely does such swelling indicate the infection site. For example, after inoculation on the hind quarters, the glands in the jaws and shoulders are often swollen, while, on the other hand, anthrax from feeding sometimes leaves the glands in the jaws unaffected and the glands in one or both inguinal regions are swollen. Changes in the lymph glands seem to be determined less by the infection site than by subcutaneous suggillation. The glands in the area of suggillation are always prominently swollen, and because suggillation occurs most frequently in the loose cell tissues of the throat, the glands near the top of the breast are more often swollen than those of the shoulders and jaw.

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    Because the glands in the jaws were frequently swollen, Pasteur concluded that infection must have occurred in the mouth. In his dissections, he apparently gave little attention to the other glands Otherwise, he would not have overlooked how they had changed, and he would probably not have arrived at his erroneous interpretation.

    Having thus established the means by which natural infection occurs, we can test the resistance to natural infection of animals that have received Pasteur's preventive inoculations.

    For this purpose, eight inoculated sheep and one uninoculated control animal were injected with reliable virulent material from spontaneous anthrax. The control animal and one sheep that had received preventive inoculations died of anthrax within two days. Because one sheep that had received preventive inoculations died, the material used for the control inoculation was virulent. This control inoculation with virulent matter can be regarded as a subsequent protective inoculation. One could expect that these animals, which withstood two preventive inoculations and, in addition, an inoculation with a very virulent anthrax substance, had arrived at the maximum level of immunity.

    Twelve days later, the remaining seven sheep and one additional uninoculated control animal were fed anthrax spores. These spores were in cultures on potatoes and were from the same anthrax material that had been used for the last inoculation. The control animal and two thrice-inoculated sheep died of anthrax within two days. Thus, the same material that killed two of eight sheep in preventive inoculations, killed two of seven sheep in the feeding tests although their immunity had been increased in the meantime by inoculation. I do not doubt that by ingesting anthrax spores, all the sheep that received Pasteur's two preventive inoculations would have become infected and died.

    Our experiment proves that Pasteur was mistaken in assuming that the natural infection of animals was less dangerous than artificial anthrax. On the contrary, the sheep were much more susceptible to natural intestinal infection than to inoculated anthrax. We have seen that to make sheep immune to Pasteur's control inoculation, twelve percent were lost in the preventive inoculation. Immunity to the stronger poison taken from local spontaneous anthrax would require losses of about twenty percent. To protect sheep against every kind of anthrax, namely against natural infection, the preventive inoculation would require matter so virulent that losses could be that much higher again.

    Our results in inoculating sheep against natural infection are in complete harmony with those obtained in Kapuvar and Packisch, where the experiments were conducted by Pasteur's own assistants and were supervised by a commission of experts. In Kapuvar and Packisch there were two trials. The first was intended to prove that preventive inoculations would immunize sheep against the virulent matter that Pasteur shipped from Paris. This trial was successful, certainly with the reservation that losses from preventive inoculations were much higher than Pasteur had expected. The second experiment was to prove that preventive inoculations protect sheep against natural infection. This trial was a failure. To test the resistance of inoculated sheep to natural infection, both commissions adopted this procedure: After inoculation, the animals were immediately placed with a similar number of uninoculated sheep in a meadow where, according to experience, anthrax was prevalent. This experiment was poorly arranged because it left too much to chance. Anthrax does not appear immediately in a herd, even if the herd is placed in an infected pasture. Moreover, individual cases are not evenly distributed through time. The disease can have long pauses and then occur in groups. By accident, anthrax could occur among uninoculated animals and not among inoculated ones. This would certainly not prove that inoculated animals were immune because there was no way to establish that all the animals were equally exposed. We found this in our feeding experiments. Thus, given this way of conducting the trial, the death of the uninoculated animals would prove little or nothing. On the other hand, the death of inoculated animals is conclusive evidence against Pasteur's theory.

    The tests in Kapuvar and Packisch went as follows: In Kapuvar between 28 September and 10 October, 267 sheep were inoculated with the first and second vaccine. Three sheep died of anthrax after the first inoculation, and ten more after the second. Of 221 control sheep that were not inoculated, one died of anthrax in the same period. Judging by the losses occasioned by the preventive inoculation, the inoculation matter was fairly strong. The 254 remaining inoculated sheep and the 220 that were not inoculated were then driven into a common pasture. According to a report in the Wiener landwirtschaftlichen Zeitung, of the inoculated sheep, two died from spontaneous anthrax and three of other diseases. Of those that were not inoculated, four died of anthrax and one died of another disease.30

    In Packisch between 10 and 20 May of this year, 251 sheep were twice inoculated, and 231 remained uninoculated. After the first inoculation, no sheep died; after the second, only one died.

    To test their immunity, twenty-four sheep that had been inoculated were inoculated again with Pasteur's virulent material. One died of anthrax in two days and another in fourteen days. This last death may not have been because of the control inoculation. From the many anthrax inoculations that I have observed, there has never been such a long period of incubation. This case may have been a result of natural infection.

    In July and August, only a few months after inoculation, three of the inoculated sheep died of anthrax. One died under circumstances resembling anthrax, but because the cadaver had decomposed, a conclusive diagnosis was no longer possible. Two died of other diseases. Eight uninoculated sheep died of anthrax. Moreover, in Packisch of eighty-three cattle that were inoculated, one died of anthrax.31

    The difference in losses between the inoculated and the uninoculated animals in both tests is insignificant, and, given the arrangement of the experiments, could be due to chance.32 Thus, there is simply no evidence of actual protection against natural infection. The tests at Kapuvar and Packisch, therefore, were decisively unfavorable to Pasteur's theory.

    The tests mentioned earlier in Beauchery and Montpothier, which Mathieu reported, led to similar results.33 In these attempts, vaccine was inoculated that Pasteur provided after 1 April of this year, and, according to his claims, it was particularly good. Thus, one cannot object that the inoculation material was too weak. In Beauchery from 25 April until 8 May of this year, 296 mutton were inoculated. Between 22 and 24 June, four mutton fell to spontaneous anthrax. During the same time, of eighty mutton that belonged to the same herd but that had not been inoculated, there were no losses. In Montpothier about one month after these three preventive inoculations, six of 203 mutton died of anthrax.

    It is remarkable that so few experiences regarding the immunity of inoculated animals to natural infection have been communicated--at least they have not been announced in such a way that they are scientifically useful. I have reported nearly everything that is known. As one sees, all the facts that are now available speak against preventive inoculation. In addition, however, there were many more unfavorable experiences. This follows from Pasteur's remarks, already cited, at the 8 June meeting of the Société centrale de médecine uétérinaire. He said that he knew of many additional failures due to the defective manufacture of the vaccine that was supplied during the winter.34

    At that time this excuse was still effective. Since then, however, the same failures have been reported after inoculations with the more powerful inoculation material that has subsequently been provided. Pasteur must also have known these facts when he gave his lecture in Geneva. In particular, he must have known of the failure in Packisch at least as well as I knew it myself. But none of this kept him from reporting only favorable results from the preventive inoculations of 250 sheep in Packisch. The favorable aspects of the test were due only to the use of vaccine that was too weak. Pasteur completely ignored the fatalities from natural infection that were known to have occurred among inoculated sheep. He also ignored all the unfavorable experiments in France, as well as the important tests in Hungary that were carried out by his assistants and were supervised by a commission.

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    Thus, Pasteur follows the tactic of communicating only favorable aspects of his experiments, and of ignoring even decisive unfavorable results. Such behavior may be appropriate for commercial advertising, but in science it must be totally rejected. At the beginning of his Geneva lecture, Pasteur placed the words "Nous avons tous une passion supérieure, la passion de uérité."  Pasteur's tactics cannot be reconciled with these words. His behavior is simply inexplicable.

    One can now judge Pasteur's methods in his preventive inoculations. If handled correctly, anthrax bacilli can be weakened and used as inoculation material against more virulent material that has also been weakened. Immunity cannot be achieved in all species of animals. As yet, Pasteur's procedure applies only to cattle and sheep. There are significant losses if one attempts to immunize the animals against natural infection. The smaller the losses from the preventive inoculation, the less protection one achieves.

    A few further circumstances are significant for the practical use of inoculation. First, one must decide how long the inoculation is effective. As yet only very inconclusive experiments have been carried out. However, Pasteur assumes that the animals are protected for approximately one year, and that they must be reinoculated annually. If this is the case, then losses incidental to injection will far surpass losses to natural infection, even in areas where anthrax is especially devastating. One must also consider the hygienic significance of preventive inoculations. In the second inoculation, one uses material that kills sheep and that is, therefore, nearly as virulent as natural anthrax poison. This slightly weakened disease poison may not be completely harmless to humans. In inoculating thousands of sheep, this poison may be widely distributed. This increases the possibility of infecting uninoculated sheep, and it may endanger humans who have contact with the wool or who eat recently inoculated animals. I recall the program for inoculating against sheep pox. This practice involved relatively few losses. The inoculations provided reliable protection, and the disease was not dangerous to humans. Nevertheless, observers concluded that the disease was maintained and spread primarily by the inoculations. This was seen as a sufficient reason to forbid inoculations.

    Therefore, because it provides inadequate protection against natural infection, because its protection does not last, and because of the danger it creates for humans and for uninoculated animals, Pasteur's preventive inoculation does not have a significant practical value. This does not mean that preventive inoculations have no future, only that because of these weaknesses, Pasteur's method is not reliable. Other improved methods may later accomplish that which one had expected of Pasteur's imperfect approach.

    Thus, as presently used, attenuated anthrax poison is of questionable value as a preventive inoculation. Yet the discovery that anthrax bacilli can be weakened and used as inoculation material has great value.

    Even before Pasteur, Toussaint found that anthrax blood lost virulence and was weakened after treatment with various agents, for example, after the addition of a one-percent solution of carbolic acid, or after warming to 55°C.35 He also found that subcutaneous injections of blood that had been altered in this way would make sheep and young dogs immune to inoculation with unweakened anthrax poison. Thus, Toussaint discovered that anthrax virus could be weakened and used for immunization. However, Toussaint's experiments were unreliable and he misconceived the operation of the attenuated virus. Toussaint assumed that anthrax bacilli had to be killed or removed from the blood. Pasteur's great service has been to prove that precisely the anthrax bacilli are the blood factors that must be changed and weakened, and that their new properties are retained by subsequent generations of bacilli. The great scientific significance of his discovery lies in this last circumstance. This provides, for the first time, an exact and unchallengeable proof that a pathogenic species of bacteria can loose its pathogenicity without having been changed morphologically. This fact is not only important for etiological exploration, but is equally interesting for biological science. It will undoubtedly point the way to other important discoveries. However, as large as Pasteur's service may have been in this valuable enrichment of science, it still seems unjust that Toussaint's name either remains completely in the background or is not even mentioned. To remain perfectly disinterested, I will cite only (Henri-Marie] Bouley, who is Pasteur's ardent disciple. In the meeting of the Academy on 8 March 1881, the same meeting in which Bouley reported Pasteur's attenuation attempts, he made the following comments: "I maintain that Toussaint had the merit of demonstrating, by a procedure that he discovered, that anthrax virus could be transformed into a vaccine virus against itself. Toussaint is the inventor of the method by which this is done. This method has proved effective, and, for the first time, has scientifically resolved the problem of the weakening of the anthrax virus and of its transformation into a vaccine virus." 38

    Pasteur also significantly improved the procedure for converting anthrax virus into inoculation material. Ultimately, from a purely scientific rather than a practical point of view, it does not matter how many animals are lost in preventive inoculations. For science, everything depends on the fact that artificial immunity can be created. However, Toussaint's procedure yielded such uncertain results that at first it seemed inconclusive. Pasteur's method, on the other hand, completely proved the success of artificial immunization.

    Nevertheless, I cannot accept Pasteur's explanation of the process by which bacilli are attenuated.

    Pasteur assumed that the influence of oxygen gradually weakened the bacilli. He used higher temperatures only to prevent spore formation. Supposedly, spores were less susceptible to the influence of oxygen. But many facts indicate that oxygen is less effective than heat and that, by metabolizing, the bacilli themselves create the substances by which they are weakened.

    In respect to the weakening influence of temperature, one can mention the following facts. Toussaint found that if anthrax blood was warmed for ten minutes at 55oC, its virulence was reduced, and that it was converted into an inoculation material. [Augusta] Chauveau further examined this observation, and recently reported his experiments.37 Moreover, the lower the temperature, the slower anthrax became. At 52oC weakening required fifteen minutes; at 50oC it required twenty minutes. As I mentioned earlier, the same circumstances were revealed by the lower temperatures that Pasteur used. In his experiments, at 43oC weakening required six days, and at 42oC it required about thirty days. [Saturnin] Arloing, [O.] Thomas and [Charles] Cornevin provided further evidence that heat weakened the bacilli.38 They found that the spores of Rauschbrand bacilli lost their virulence and became a Rauschbrand vaccine if they were warmed for six hours at more than 85oC. Finally, [A.] Fitz heated the spores of Bacillus butyricus (the ferment of butyric acid).39 If the bacilli were heated to 90oC for five hours, or to 80oC for seven hours, they were still able to propagate but no longer caused fermentation. In these last two cases, in which the high temperature operated on spores, the influence of oxygen was totally excluded. Warmth was the only agent that could enter into consideration.

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    In addition to heat, other factors can weaken bacteria. Toussaint also provided the first clues in this area. He showed that the virulence of anthrax blood could be reduced by the addition of carbolic acid. Carbolic acid--phenol--is known to be a product of bacterial metabolism. Many other facts, which I cannot enumerate in this context, suggest that other substances produced in the growth and propagation of bacteria also weaken and retard the bacteria by which they are produced. The more slowly anthrax bacilli are weakened and the more time they are allowed to grow and to propagate, the more such metabolic by-products weaken them.

    To support his theory that oxygen weakened bacilli, Pasteur noted that anthrax bacilli retained their virulence if warmed to 42o or 43oC in the absence of oxygen, but that they lost virulence if oxygen was present. But Pasteur forgot that in the absence of oxygen, anthrax bacilli could not grow, and that they could not, therefore, create the metabolic by-products that reduce their virulence.

    The following facts prove conclusively that oxygen is not the weakening influence. If one repeatedly inoculates new culture media without excessive intervals between, its characteristic virulence remains unchanged. However, if one leaves the bacilli in the same medium without further nourishment, and if they do not produce spores, their virulence gradually declines and can ultimately be lost. Oxygen is equally present in both cases. Yet the bacilli are weakened in one case and not in the other. Provisionally, I would give the following explanation. Bacilli that are not further cultured and that remain constantly in contact with the products of their own metabolism become weakened by those products. Bacilli that are repeatedly transferred into fresh media are removed from the detrimental influences and remain unweakened. Yet oxygen influences both, and all other conditions remain the same.

The process of attenuation cannot be as simple as Pasteur imagines. To satisfactorily explain this process, one must consider various different factors such as warmth, the operation of chemical agents, and almost certainly other conditions that are as yet unknown.

    I have a few general concluding remarks to make about the attenuation of disease material.

    In Pasteur's opinion, four different infection materials have already been attenuated. On the basis of this success, in his opinion, one can assume the operation of a universal law of attenuation and of conversion into protective inoculation material. Supposedly, such a law is binding on all pathogenic organisms. In my opinion, one cannot go this far. As yet, only the attenuation of anthrax is an uncontested fact. For the other infection materials that Pasteur includes, namely for microbes of chicken cholera, the nouvelle maladie de la rage, and rabbit typhoid, the critical examination and confirmation of reliable observers is still missing. This defect is all the more important since two of these diseases are unquestionably identical to rabbit septicemia. Moreover, according to Toussaint's investigation, chicken cholera may be the same disease as rabbit septicemia. Because I have mistakenly been identified as a principal opponent of the transformation of pathogenic microorganisms, I will repeat what I said in a communication from the Gesundheitsamt and on numerous other occasions. "I do not, in principle, oppose the theory that one species can be transformed into another closely related species. I do not regard it as impossible that a pathogenic organism could be converted into a harmless one or vice versa. Everyone will agree that if certain conditions were to bring about such a change, this would have exceptionally important consequences. For this very reason, science can fully recognize such an occurrence only when it is proved exactly and in a way that precludes all doubts."40  I am still of the same opinion, and now, since an exact proof of the transformation of anthrax bacilli has been given, I regard it as an established fact. However, I require equally uncontrovertable proof for other transformation experiments. Before one can talk of a law of transformation for pathogenic microorganisms, one must transform many of them. Researchers in this area must be more objective and self-critical. Also, after the experiments that have been discussed, one must warn against hasty practical applications of scientific results. The hopes aroused by Pasteur's inoculations for chicken cholera have apparently not been fulfilled. At least chicken owners are not inoculating with the weakened microbes of chicken cholera. For the time being, preventive inoculations for anthrax seem to be equally devoid of practical value. So far, no actual successes can be cited for them. Thus, while Pasteur may have been celebrated in the Geneva Congress as a second Jenner, the celebration was somewhat premature. In the surge of enthusiasm, one forgot that Jenner's discovery had value for humans rather than for sheep.

    We already know a range of different bacteria that attack humans directly; these include tuberculosis, leprosy and abdominal typhoid bacilli, erysipelas micrococci, and relapsing fever spirochaeta. Only if we succeed in weakening these and in converting them into protective inoculation material, will preventive inoculations with weakened infection material truly be a triumph worthy of celebration.

NOTES       (personal pronouns refer to Professor Codell)

1. [Louis Pasteur, "De l'atténuation des virus," Congres international d'hygiène et de démographie, Comptes rendue 1882, Genève, H. Georg, 1883, vol. 1, pp. 127-145. ]
2. Ibid., p. 145. Koch's editor, Julius Schwalbe, quotes Koch's protest, see above, Introduction, note 1, vol. 1, pp. 207f.]
3. [By the time this paper was delivered, Koch's first paper on tuberculosis had been published.]
4. [Friedrich A. Fehleisen, Die Aetiologie des Erysipelas, Berlin, T. Fischer, 1883, pp. 1-35.1
5. [George Miller] Sternberg,
[A Fatal Form of Septicemia in the Rabbit Produced by the Subcutaneous Injection of Human Saliva, Baltimore, John Murphy, 1881, reprinted from the] National Board of Health Bulletin, 30 April 1881; Edme-Felix-Albert Vulpian, Bulletin de l'Académie de médecine, 29 March 1881, [2nd series, 10:394f].
6. [H. Toussaint, "Identité de la septicémie expérimentale aiguë et du choléra des poules," Comptes rendues de l'Académie des sciences, 1880, 91:301303.]
7. [Davaine, see above, p. 53, note 5; Gaffky, see above, p. 80, note 18.]
8. [Sternberg, op. cit., note 5 above.]
9. [Wilhelm Stuck, "Vorläufige Mittheilung ü1ber die Arbeiten des kaiserlichen Gesundheitsamt welche zur Entdeckung des Bacillus der Rotzkrankheit gefüihrt haben," Deutsche medizinische Wochenschrift, 1882, 8:707f.]
10. [Obidou Lannelongue and Maurice Raynaud, "Recherches expérimentales sur la transmission du virus rabique de l'homme au lapin, Bulletin de l'Académie de médecine, 1881, 2nd series, 10:61-84; Louis Martineau and Paul Hamonic, "De la bactérie syphilitique; de 1'evolution syphilitique chez le pork," Union médicale, 1882, 3rd series, 34:398-403.1
11. Annales d'hygi
éne publique [et de Médecine légale, 1882, 8:]301 f.
12. [H. Oemler, "Experimentelle Beiträge zu Milzbrandfrage," Archiv fur wissenschaftliche and praktische Thierheilkunde, 1879, 5:164-212.]
13. [Friedrich Löffler, "Zur Immunitätsfrage," Mittheilungen aus dem Kaiserlichen Gesundheitsamte, see above, p. 80, note 18.]
14. [Alfredo Gotti, "Supra alcuni experimenti di inoculazione carbonchiosa preservative nei bovini," Accademia delle scienze dell'Instituto di Bologna, Memorie, 1883, 4th series, 5:734-738.]
15. [Alfred Guillebeau, "Die Schutzimpfung gegen Milzbrand," Schweizer Archiv für Tierheilkunde,
1882, 24:129.]
16. [I have not been able to locate Koch's source for this reference.]
17. [Report of transactions of the 8 June 1882 meeting, Bulletin de la societé centrale de médecine vét
érinaire, 1881-1883, 4th series, 5-6:267-274.]
18. [Löffler, op. cit., note 13 above.]
19. [I have not been able to locate Koch's source for this reference.]
20. [Robert Koch, Georg Gaffky and Friedrich Löffler, "Experimentelle Studien üiber die künstliche Abschwächung der Milzbrandbazillen and Milzbrandinfektion durch Fütterung," Schwalbe, see above, Introduction, note 1, vol. 1, pp. 232-270. ]
21. [See note 16 above.]
22. [I have not been able to locate Koch's source for this reference.]
23. [P. Börner, "Pasteur's Schutzimpfung gegen den Milzbrand vor der deutschen Kritik,"] Deutsche medizinische Wochenschrift, 1882, [8:697f, Müller, "Die Milzbrandimpfungen in Prakisch,"] Archiv fur wissenschaftliche and praktische Tierheilhunde, [1882,] 8(:319-338].
24. Mathieu, ["Sur la vaccination charbonneuse," Bulletin de la] Societ
é centrale de rnédecine uétérinaire [1881-1883, 4th series, 5-6:305-309].
25. Taken from a personal communication.
26. [I have not been able to identify Koch's source for this reference.]
27. [Op. cit., note 17 above, p. 269.]
28. [See note 26 above.]
29. [Mathieu, "Sur la vaccination charbonneuse,"] Recueil de m
édecine uéetérinaire, 1882, 6th series, 9[:796-800].
30. [Koch cites] Wiener landwirtschaftliche Zeitung, 27 August 1883 (but I have not been able to verify this reference].
31. Archiv für wissenschaftliche and prahtische Tierheilkunde, (1882,) 8:[468-479,] p. 468.
32. According to a communication I received, another of the inoculated sheep in Packisch recently died of anthrax. At this point, therefore, six inoculated and eight uninoculated sheep have died from natural infection.
33. (Mathieu, op. cit., note 24 above.]
34. [Op. cit., note 17 above.]
35. [H. Toussaint, "De l'immunit
é pour Ie charbon acquise à la suite d'inoculations preventives," Comptes rendus de l'Académie des sciences, 1882, 94:310f.]
36. (Henri-Marie Bouley, this was a remark made in a discussion reported in Bulletin de l'Acad
émie de médecine, 1881, 10:301.]
37. [Augusta Chauveau, "Etude exp
érimentale des conditions qui permettent de rendre usuel 1'emploi de la méthode de M. Toussaint pour atténuer Ie virus charbonneux," Comptes rendus de l'Académie des sciences, 1882, 94:1,694-1,698.]
38. [Saturnin Arloing, 0. Thomas, and Charles Cornevin, "Note relative à la conservation et à
la destruction de la virulence du microbe du charbon symptomatique," Recueil de médecine vétérinaire, 1882, 9:467-472, 762-764.]
39. [I have not been able to identify Koch's source for this reference.]
40. [See above, p. 76.]

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