- Email: [email protected]
Investigations Regarding the Production of Diphtheria Immunity in Animals
GENERAL ARTICLES.
35
This cow had no glandular enlargements, and physical examination of the chest revealed n\levidence of lung consolidation. It.was therefore concluded, relying upon the absence of reaction to Koch's fluid, that possibly the animal was the subject of some non-tuberculous lung affection. The animal was killed on the 2Sth February, and the postmortem revealed most extensive tuberculous lesions. The lung was the seat of scattered patches of tuberculous catarrhal pneumonia, the bronchial glands were greatly enlarged, and both parietal and visceral pleura and peritoneum were literally covered with perlsucltt nodules. Not the slightest trace of Koch's fluid having excited any reaction in the tuberculous parts could be detected. Had the experiments with this cow stood alone, it might have been urged that the non-reaction was due simply to the dose of Koch's fluid having been too small. But the experiments with the first cow showed that even 20 milligrammes sufficed to elicit a decided reaction, whereas the second cow received in the third experiment So milligrammes. As further evidence that the result was not ascribable to the smallness of the dose, the experiments recently published by Dr Stricker l may be cited. In these experiments I ccm. of a I °1o solution sufficed to determine in four tuberculous cows a rise of temperature, amounting in one of the animals to lOS·So. Only a few experiments with Koch's fluid on cattle have yet been published, but in almost all it is claimed that a reaction has been obtained in the tuberculous animals, while in no case has there been any rise of temperature in the control non-tuberculous animals. It is therefore permissible to hope that there has been discovered a substance that will enable veterinary surgeons to diagnose tuberculosis in dairy cattle with greater precision than has hitherto been possible, but the result obtained with the second cow above referred to proves that difficulty of diagnosis may still exist, even in cases where the lesions are very extensive. In all the experiments yet published the tuberculous animals appear to have been in a rather advanced stage of the disease, but it still remains to be proved whether any discernible reaction will follow the injection of Koch's fluid when the lesions are of small extent. [In the foregoing experiments the observations regarding the pulse, temperature, etc., were made by Mr Mettam, M.R.C.V.S., clinical assistant, Mr Stewart Stockman, M.R.C.V.S., and myself, assisted by relays of students.]
INVESTIGATIONS REGARDItiG THE PRODUCTION OF DIPHTHERIA IMMUNITY IN ANIMALS.2 By Stabsarzt Dr BEHRING. KITASATO and I have already reported experiments which prove that the immunity of the hitherto examined animals against tetanus depends upon the power of their blood to render the poisonous substances produced by the tetanus bacilli innocuous. 1 Stricker's Archiv f. animalische Nahrungkunde, ~o. 4, and Oester. l\Ionatschrilt fur Thiel'heilkunde, March 189], 2 Translated from the Deutsche lIIed. Wochenschrift 1890, No. 50.
GENERAL ARTICLES.
In that article the same was asserted with regard to diphtheria immunity, but the particular experiments which had proved that the mechanism was the same in both diseases were not reported. To make good that deficiency is the object of the following communication. As was first proved by Lomer, and then by Roux and Yersin, there are animals that are by nature immune against diphtheria. Through personal investigation I was able to ascertain that mice and rats belong to this class, and that these animals could bear, without recognisable disturbance of health, inoculation with cultures, in quantities that were certainly fatal to much larger animals, such as guinea-pigs, rabbits, and sheep. Of a bouillou diphtheria culture obtained from a diphtheritic membrane of a child that had died from diphtheria in January of this year, '05 ccm. sufficed to kill guinea-pigs after 3-4 days; '03 ccm. subcutaneously injected to rabbits killed these animals after 2-4 days; and 2 ccm. proved fatal for an adult sheep after 50 hours. I injected '3 ccm. of the same culture into mice, without producing any striking symptoms of disease in these animals. It is further possible to render immune animals that originally are very susceptible to diphtheria, and, indeed, there are very various ways of attaining this result. I. One of the methods of producing immunity, which I can on the ground of my own experiments assert to be very sure, has been acc,urately described by Professor C. Frankel in the Berliner klinisclte Wochenscltrift " it consists in the use of sterilised cultures. and by means of this one is in a position in 10- I4 days to make guinea-pigs insusceptible to inoculations that are certainly fatal to normal guineapigs. ' 2. Further, I have made guinea-pigs immune in the following manner. I added to four-weeks-old cultures trichloride of iodine in such quantities that the same was contained in the culture in the proportion of 1 to 500, and I allowed the trichloride of iodine to act on the culture for sixteen hours, I then injected 2 ccm. of the culture thus treated into the peritoneal cavity of two guinea-pigs. After three weeks I infected the guinea-pigs with 2 ccm. of a diphtheria culture which had grown for four days in bouillon with 1 : 5500 of trichloride of iodine added. The control animal died after seven days; both the previously treated animals remained alive. . After other fourteen days the two animals resisted as much of a fully virulent diphtheria culture as sufficed to kill normal guinea-pigs after thirty-six' hours. In both the methods just mentioned the immunity is brought about by the products generated in the cultures by the metabolism of the diphtheria bacilli. 3. But it is possible to produce immunity also by means of those products of metabolism that are generated in the organism of the living animal by the diphtheria bacilli. In animals dead from diphtheria, there is very frequently found in the pleural cavity an amber yellow, but sometimes colourless, and in other cases bloody, transudate. The quantity of the same is very variable. Generally in guinea-pigs it amounts to not more than 1-5 ccm., but in not seldom"
GENERAL ARTICLES.
37
cases I have been able to obtain up to IS ccm. from a single guineapig. 111 more than fifty different cases examined this transudate never contained diphtheria bacilli, but it possesses toxic properties for guinea-pigs. The degree of toxicity is not always the same, and I have gathered the impression that the bloody transudate is more poisonous than the light coloured. But even after subcutaneous and intra-abdominal injection of, on an average, 10- IS ccm. of non-bloody transudate, the majority of the guinea-pigs died after a few (up to ten) days; at the post-Jl~orte11l one finds h
GENERAL ARTICLES.
received I. per cent., two larger 2 per cent. solution) subcutaneously injected at the seat of infection all remained alive; in two animals, the treatment was first begun after six hours, and one of the same died after four days, while the other remained alive; all the animals received on the three days immediately following a new injection of trichloride of iodine. I have not been able to obtain at all certain results beyond six hours after infection, even when the animals were so slightly inoculated that normal animals did not die: until after four days. The surviving guinea-pigs are for a long time ill. Their recovery, is ushered in by a demarcating inflammation; later a dry scab forms, and this steadily becomes less adherent, until ultimately one can lift it' off. Under this scab, living and virulent diphtheria bacilli have been recognisable at the end of three weeks. Jf one now infects such animals, whose general condition has become quite good, but in which an open ulcerating surface still exists, they show a decidec,1ly greater power of resistance towards infection than the normal; however, it was only after complete healing and cicatrisation that I found several animals which had been cured with trichloride of iodine (and Dr Boer one cured with auro-chloride· of sodium) immune to such a degree that they resisted inoculation with highly virulent diphtheria which was fatal to the control animals after thirty-six hours. I may remark in passing that one can obtain better curative results with trichloride .of iodine in the case of rabbits. These animals could be cured without their acquiring a scab at the seat of inoculation, and treq.tment was successful even after twenty-four hours, when the infection had been of such a strength that the control animals died in four days. Whether the recovered rabbits possess any immunity, I ani not yet in a position to say. I take this opportunity to prevent any mistake as to whether we possess in trichloride of iodine, which is in a position to call forth such a decided therapeutic action in animals, a remedy for diphtheria in man. Apart from the corrosive action of this substance, and apart from the fact that I have made only a few observations concerning the possibility of cure in animals that have been infected from the larynx or trachea, special cautious experiments made on children suffering from diphtheria have not afforded 'much encouragement for the application of trichloride of iodine, and I declare that I have no remedy for human diphtheria, but I am searching for it. As regards the production of diphtheria immunity through the above-sketched methods, I was induced to adopt the view of an action on the part of the products of metabolism of the diphtheria bacilli, through the fact that I had succeeded in rendering guinea-pigs immune simply by previous treatment with trichloride of iodine. 5. But we are now in a position to confer upon guinea-pigs a higher power of resistance towards diphtheria, and to protect rabbits against an otherwise fatal quantity of culture, by means of a substance that has nothing to do with the products of metabolism of the diphtheria bacilli-namely, peroxide of hydrogen. I used this substance in slightly acid (through SUlphuric acid) 10 per cent. solution, by which means it preserves its activity almost unchanged for some weeks. Guinea-pigs bear up to 2 ccm. and more
GENERAL ARTICLES.
39
of this solution-reckoned by body-weight from about I : 4000 to I : 2500; mice'l to "75 ccm. (from I : 2000 to I : 800); rabbits, on the other hand, are extraordinarily less resistant towards peroxide of hydrogen; several medium-sized animals died in less than one hour after the subcutarteous injection of I ccm. (I : 15,000 of body-weight). Altogether, the individual resisting power towards this substance varies much more than that towards anyone of more than a hundred other chemicals that I have tested. For certain cases peroxide of hydrogen is a distinguished disinfecting substance, and I originally used it with the intention of exercising a therapeutic action in diphtheria. I observed, however, that animals treated with peroxide of hydrogen after infection succumbed to diphtheria much quicker than the control animals; and as I added this substance to the diphtheria cultures in quantities approaching the fatal dose, I found the virulence of such cultures considerably heightened. On the other hand, when I some days later infected animals previously treated with peroxide of hydrogen, they had acquired a pronounced degree C?f immunity, which in guinea-pigs was shown, among other things, by the fact that there was not observable at the seat of infection very diffuse <:edema spreading to the surrounding parts, but a firm tumour tending to suppuration. On cutting into this there flowed out a clear serous fluid, which was enclosed by a dense envelope. The experiments with peroxide of hydrogen are still being pursued, and I cite here only one series of them, which, according to a common project, have been carried out in this institute by Herr Stabsarzt Ltibbert. Each of five rabbits received on the 11th, 12th, 14th, and 17th November 5 ccm. of peroxide of hydrogen. On the 20th November, in presence of Ltibbert, I injected subcutaneously '5 ccm. of perrectly virulent diphtheria culture in bouilloll to these five animals, and at the same time to a fresh rabbit. The control animal died of typical diphtheria on the 21st November, that is, after twenty-four hours. Of the previously treated animals one died on the 25th November (after five days), two on the 27th November (after seven days), one on the 28th November (after eight days), and one showed no symptom of illness and has remained healthy. Of the five different modes of immunity against diphtheria previously mentioned, the last four belong to those which Pasteur first taught us. The fifth, which brings about immunity by substa,nces quite foreign to the agents that excite the infection-indeed, of a perfectly simple chemical nature. has as yet scarcely any analogy. That the possibility of producing immunity by simple chemical substances exists also in the case of other infectious diseases can be proved by the' fact which. through agreement with Herr Kitasato, I here report, namely, "that one of the metheds of rendering rabbits immune against tetanus consists in previously treating them exclusively with trichloride of iodine solution." The whole five of the already-described methods' of producing immunity against diphtheria are, at least in the form in which I here report them, in my opinion not applicable to the human subject. But from a scientific point of view, and especially for the solution of
GENERAL ARTICLES.
the problem with which alone I am here concerned-for the comprehension of the production of immunity against diphtheria, they are capable of rendering valuable service. However the immunity may be brought about, and I do not exclude the natural immunity against diphtheria, alI diphtheria-immune animals have in common certain properties which distinguish them from non-immune animals. In the first place, alI living immune animals are protected, not only against infection with the living baciIIi, but also against the deleterious action of those poisonous substances which are formed in cultures and in the animal body by diphtheria bacilli. The investigation of this I have carried out in different ways. In the first place, I experimented with the solution of an albuminous substance which I separated from old cultures by means of acid alcohol; I was, however, unable to remove the acid from the preparation obtained without injury to the action of the poison. I also found it no easy task to separate from the precipitate ol::itained other accessory substances, although the earlier investigators in this sphere succeeded without difficulty. But for the present purpose it was not necessary to separate the diphtheria poison-ot, perhaps it would be more correct to say, the diphtheria poisons., Filtered old cultures afforded me alI that I wanted. I found that after ten weeks my cultures grown in alkaline bouilloll with 10 ccm. normal alkaline solution per litre, contained so much poisonous substance that, when made germ-free by filtration, a dose of I ccm. produced in medium-sized guinea-pigs characteristic symptoms of diphtheria poisoning, which entirely disappeared only after 3-4 weeks. From 3-4 ccm. suffice to kill even larger guinea-pigs after 3-8 days. Necrosis of the skin occurred very regularly, and, indeed, not simply at the seat of infection, but quite remote from thatmost frequently on the belly. AlI the guinea-pigs which now possessed a firm diphtheria immunity-that is such as no longer showed any symptoms of illness even after repeated infection-bore 3-5 ccm. without exhibiting any symptoms of poisoning or any local reaction whatever. On the other hand, the guinea-pigs not quite recovered from an infection showed' themselves only very little more resistant to the poison than normal animals, and necrosis of the skin appeared in the same manner as in the latter. It is very remarkable that the immunity can again be lost in consequence of the subcutaneous injection of large and repeated doses of the poison; this occurs the more certainly, the less firmly fixed the immunity has been. In every case the guinea-pigs under the influence of the poisonous germ-free diphtheria culture are in less favourable conditions towards diphtheria infection than before. One might at first suppose that the resistance to poison here described depends upon a "poison habit," such as is alIeged to exist in the case of drunkards, opium eaters, and arsenic eaters, certain vital centres ceasing to react to the particular poison-in short that it is in reality a case of accustoming or inuring of the vital organs. But such a view is here immediately opposed to the fact that even such animals as never had anything to do with the diphtheria poison possess the power of resistance to that poison. To return again to the germ-free ten-weeks-old culture, that is,
GENERAL ARTICLES.
41
reckoned by body-weight, fatal to guinea-pigs in the proportion of about I: roo; but mice stand the poison without injury when it is injected into them on the proportion of I : 20; and I have injected into rats 4 ccm. on several successive days without setting up any reaction worth naming. Opposed to the view of a poison habit there is the further fact that I could not succeed by the most careful ascent from quite innocuous to higher doses of the poison in protecting the animals against the diphtheria poison so as t<;> make them ,subsequently tolerate a little more of it than normal animals. These observations and reflections led me to consider more closely whether the cause of the power of resistance to the poison may depend, not on a peculiarity of the living cellular parts of the organism, but upon a special peculiarity of the blood, apart from living cells. In order to decide this question, I took blood from rats into the peritoneal cavity of which large quantities of the diphtheria poison had been introduced three hours previously, and injected this blood or the serum obtained from it into the peritoneal cavity of guinea-pigs; no symptoms of poisoning appeared in the latter, whereas the blood of animals susceptibJe to diphtheria, and which had received the diphtheria poison in the same quantity into the peritoneal cavity, while it did not kill guinea-pigs, at least made them distinctly ill. Further, I attacl;1 importance to the fact that even the extravascular blood of guinea-pigs immune against diphtheria possesses the power to render the diphtheria poison innocuous. To what degree this is the case, and to what extent therapeutic results may be obtained with the blood of animals rendered immune, I intend to consider in a later communication. I have still to mention a negative criterion common to immune animals. Before I had arrived at those investigations which enabled me to find recognisable distinctions between the blood of animals susceptible to diphtheria and that of those immune against the disease, I had also employed the methods hitherto in use in examining blood. I had in this way examined the blood of four guinea-pigs and two rabbits that had been rendered immune, as well as the blood of over ten rats, as to whether there could be recognised in it any influence adverse to bacteria. Such was not the case; in all cases the diphtheria bacilli grew very luxuriantly, and their virulence was rather increased. After I had made these observations in my investigations concerning the production .of diphtheria immunity, Herr Kitasato and I in common applied them to the case of tetan.us; and in our first communication we have succeeded, as we think, in furnishing the indisputable proof that in tetanus the poison-destroying action of the blood of tetanus-immune animals is a causa sujjiciens for the production of the immunity. The fortunate circumstance that we could obtain from an immune rabbit so much blood and serum with which to treat a larger number of the much smaller mice, has made it possible to expose the therapeutic consequences fully and completely. It is possible that in my article published in common with Kitasato the curative effect of the transfusion of blood from tetanus-immune rabbits was not expressed with sufficient distinctness, and I will therefore here give special
GENERAL ARTICLES.
prominence to the statement that mice are not merely rendered imll?-uQe, I;>y the blood of tetanus-immune rabbits, and that the protection of mice is brought about not m(C:rely when blood or serum ,of immune animals is injected into their peritoneal cavity immediately after infection, and before they have contracted tetanus; even }vhen severa! extremities have already becom~ tetanic, and when, accorqing, to previous experience, they might be expected to die in a few, hours if not treated-even then a cure can be effected with great c,ert!linty, and, indeed, so rapidly that within a few days no trace of disease is observable. The,possibility of a cure in the case of diseases with a quite acute course can therefore no longer be denied.
ON EXTRA-UTERINE PREGNANCY IN THE CAT. By D.
J.
HAMILTON, M.B., Professor of Pathology,
Aberdeen University.
QUITE apart from the practical issues bound up with it, the subject of extra-uterine pregnancy considered from the purely biological point of view is one of great interest. The case which I am about to describe occurred in a cat, and is worth recording from extra-uterine pregnancy being a somewhat rare condition in the lower animals, as well as from the unique attachments which the progeny had contractedwith neighbouring parts. It is asser,ted that there are three situations in which primarily the embryo or embryos may be found; Firstly, occupying a Fallopian tube; secondly, within the ovary itself; and, t/lirdly, growing from the peritoneum. The great majority of extra-uterine pregnancies, in Man at least, are acknowledged on all hands to be tubal in their origin. The fcetus grows within the tube until about the end of the second or the beginning of the third month. It then bursts forth in one of two directions, either into the peritoneum or into the loose cellular tissue between the layers of the broad ligament. In the case of the fcetus escaping into the peritoneal sac, which is the commoner of the two occurrences, the parent dies sooner or later if unrelieved, and usually from abdominal ha:-morrhage. The fcetus, however, on rupturing into the abdomen need not im1llediate!.y perish, nor need the condition prove at once fatal to the mother. On the contrary, the embryo may continue to grow in the peritoneal cavity until full time is reached, the placenta remaining adherent to the Fallopian tube, and the umbilical cord escaping through the rupture in its wall. The lacerated edges of the wound cicatrise, so that if examined some time after the accident has happened the cord is found to issue through an aperture with a regular clean cut margin (Sa Chiari's case, Zcitscltrift fltr Heilkunde, vol. viii., 1887, p. 127). Although the tube usually ruptures, yet it has been alleged that in some instances it goes on stretching up to the full term of gestation. A case recorded by Chiari (loc. cit., p. 128) appeared to have been of this nature. The child was fully nourished and was found lying in a sac supposed to be the distended Fallopian tube. A layer of muscular fibre lined the sac, said to be that of the tube in a state of
35
This cow had no glandular enlargements, and physical examination of the chest revealed n\levidence of lung consolidation. It.was therefore concluded, relying upon the absence of reaction to Koch's fluid, that possibly the animal was the subject of some non-tuberculous lung affection. The animal was killed on the 2Sth February, and the postmortem revealed most extensive tuberculous lesions. The lung was the seat of scattered patches of tuberculous catarrhal pneumonia, the bronchial glands were greatly enlarged, and both parietal and visceral pleura and peritoneum were literally covered with perlsucltt nodules. Not the slightest trace of Koch's fluid having excited any reaction in the tuberculous parts could be detected. Had the experiments with this cow stood alone, it might have been urged that the non-reaction was due simply to the dose of Koch's fluid having been too small. But the experiments with the first cow showed that even 20 milligrammes sufficed to elicit a decided reaction, whereas the second cow received in the third experiment So milligrammes. As further evidence that the result was not ascribable to the smallness of the dose, the experiments recently published by Dr Stricker l may be cited. In these experiments I ccm. of a I °1o solution sufficed to determine in four tuberculous cows a rise of temperature, amounting in one of the animals to lOS·So. Only a few experiments with Koch's fluid on cattle have yet been published, but in almost all it is claimed that a reaction has been obtained in the tuberculous animals, while in no case has there been any rise of temperature in the control non-tuberculous animals. It is therefore permissible to hope that there has been discovered a substance that will enable veterinary surgeons to diagnose tuberculosis in dairy cattle with greater precision than has hitherto been possible, but the result obtained with the second cow above referred to proves that difficulty of diagnosis may still exist, even in cases where the lesions are very extensive. In all the experiments yet published the tuberculous animals appear to have been in a rather advanced stage of the disease, but it still remains to be proved whether any discernible reaction will follow the injection of Koch's fluid when the lesions are of small extent. [In the foregoing experiments the observations regarding the pulse, temperature, etc., were made by Mr Mettam, M.R.C.V.S., clinical assistant, Mr Stewart Stockman, M.R.C.V.S., and myself, assisted by relays of students.]
INVESTIGATIONS REGARDItiG THE PRODUCTION OF DIPHTHERIA IMMUNITY IN ANIMALS.2 By Stabsarzt Dr BEHRING. KITASATO and I have already reported experiments which prove that the immunity of the hitherto examined animals against tetanus depends upon the power of their blood to render the poisonous substances produced by the tetanus bacilli innocuous. 1 Stricker's Archiv f. animalische Nahrungkunde, ~o. 4, and Oester. l\Ionatschrilt fur Thiel'heilkunde, March 189], 2 Translated from the Deutsche lIIed. Wochenschrift 1890, No. 50.
GENERAL ARTICLES.
In that article the same was asserted with regard to diphtheria immunity, but the particular experiments which had proved that the mechanism was the same in both diseases were not reported. To make good that deficiency is the object of the following communication. As was first proved by Lomer, and then by Roux and Yersin, there are animals that are by nature immune against diphtheria. Through personal investigation I was able to ascertain that mice and rats belong to this class, and that these animals could bear, without recognisable disturbance of health, inoculation with cultures, in quantities that were certainly fatal to much larger animals, such as guinea-pigs, rabbits, and sheep. Of a bouillou diphtheria culture obtained from a diphtheritic membrane of a child that had died from diphtheria in January of this year, '05 ccm. sufficed to kill guinea-pigs after 3-4 days; '03 ccm. subcutaneously injected to rabbits killed these animals after 2-4 days; and 2 ccm. proved fatal for an adult sheep after 50 hours. I injected '3 ccm. of the same culture into mice, without producing any striking symptoms of disease in these animals. It is further possible to render immune animals that originally are very susceptible to diphtheria, and, indeed, there are very various ways of attaining this result. I. One of the methods of producing immunity, which I can on the ground of my own experiments assert to be very sure, has been acc,urately described by Professor C. Frankel in the Berliner klinisclte Wochenscltrift " it consists in the use of sterilised cultures. and by means of this one is in a position in 10- I4 days to make guinea-pigs insusceptible to inoculations that are certainly fatal to normal guineapigs. ' 2. Further, I have made guinea-pigs immune in the following manner. I added to four-weeks-old cultures trichloride of iodine in such quantities that the same was contained in the culture in the proportion of 1 to 500, and I allowed the trichloride of iodine to act on the culture for sixteen hours, I then injected 2 ccm. of the culture thus treated into the peritoneal cavity of two guinea-pigs. After three weeks I infected the guinea-pigs with 2 ccm. of a diphtheria culture which had grown for four days in bouillon with 1 : 5500 of trichloride of iodine added. The control animal died after seven days; both the previously treated animals remained alive. . After other fourteen days the two animals resisted as much of a fully virulent diphtheria culture as sufficed to kill normal guinea-pigs after thirty-six' hours. In both the methods just mentioned the immunity is brought about by the products generated in the cultures by the metabolism of the diphtheria bacilli. 3. But it is possible to produce immunity also by means of those products of metabolism that are generated in the organism of the living animal by the diphtheria bacilli. In animals dead from diphtheria, there is very frequently found in the pleural cavity an amber yellow, but sometimes colourless, and in other cases bloody, transudate. The quantity of the same is very variable. Generally in guinea-pigs it amounts to not more than 1-5 ccm., but in not seldom"
GENERAL ARTICLES.
37
cases I have been able to obtain up to IS ccm. from a single guineapig. 111 more than fifty different cases examined this transudate never contained diphtheria bacilli, but it possesses toxic properties for guinea-pigs. The degree of toxicity is not always the same, and I have gathered the impression that the bloody transudate is more poisonous than the light coloured. But even after subcutaneous and intra-abdominal injection of, on an average, 10- IS ccm. of non-bloody transudate, the majority of the guinea-pigs died after a few (up to ten) days; at the post-Jl~orte11l one finds h
GENERAL ARTICLES.
received I. per cent., two larger 2 per cent. solution) subcutaneously injected at the seat of infection all remained alive; in two animals, the treatment was first begun after six hours, and one of the same died after four days, while the other remained alive; all the animals received on the three days immediately following a new injection of trichloride of iodine. I have not been able to obtain at all certain results beyond six hours after infection, even when the animals were so slightly inoculated that normal animals did not die: until after four days. The surviving guinea-pigs are for a long time ill. Their recovery, is ushered in by a demarcating inflammation; later a dry scab forms, and this steadily becomes less adherent, until ultimately one can lift it' off. Under this scab, living and virulent diphtheria bacilli have been recognisable at the end of three weeks. Jf one now infects such animals, whose general condition has become quite good, but in which an open ulcerating surface still exists, they show a decidec,1ly greater power of resistance towards infection than the normal; however, it was only after complete healing and cicatrisation that I found several animals which had been cured with trichloride of iodine (and Dr Boer one cured with auro-chloride· of sodium) immune to such a degree that they resisted inoculation with highly virulent diphtheria which was fatal to the control animals after thirty-six hours. I may remark in passing that one can obtain better curative results with trichloride .of iodine in the case of rabbits. These animals could be cured without their acquiring a scab at the seat of inoculation, and treq.tment was successful even after twenty-four hours, when the infection had been of such a strength that the control animals died in four days. Whether the recovered rabbits possess any immunity, I ani not yet in a position to say. I take this opportunity to prevent any mistake as to whether we possess in trichloride of iodine, which is in a position to call forth such a decided therapeutic action in animals, a remedy for diphtheria in man. Apart from the corrosive action of this substance, and apart from the fact that I have made only a few observations concerning the possibility of cure in animals that have been infected from the larynx or trachea, special cautious experiments made on children suffering from diphtheria have not afforded 'much encouragement for the application of trichloride of iodine, and I declare that I have no remedy for human diphtheria, but I am searching for it. As regards the production of diphtheria immunity through the above-sketched methods, I was induced to adopt the view of an action on the part of the products of metabolism of the diphtheria bacilli, through the fact that I had succeeded in rendering guinea-pigs immune simply by previous treatment with trichloride of iodine. 5. But we are now in a position to confer upon guinea-pigs a higher power of resistance towards diphtheria, and to protect rabbits against an otherwise fatal quantity of culture, by means of a substance that has nothing to do with the products of metabolism of the diphtheria bacilli-namely, peroxide of hydrogen. I used this substance in slightly acid (through SUlphuric acid) 10 per cent. solution, by which means it preserves its activity almost unchanged for some weeks. Guinea-pigs bear up to 2 ccm. and more
GENERAL ARTICLES.
39
of this solution-reckoned by body-weight from about I : 4000 to I : 2500; mice'l to "75 ccm. (from I : 2000 to I : 800); rabbits, on the other hand, are extraordinarily less resistant towards peroxide of hydrogen; several medium-sized animals died in less than one hour after the subcutarteous injection of I ccm. (I : 15,000 of body-weight). Altogether, the individual resisting power towards this substance varies much more than that towards anyone of more than a hundred other chemicals that I have tested. For certain cases peroxide of hydrogen is a distinguished disinfecting substance, and I originally used it with the intention of exercising a therapeutic action in diphtheria. I observed, however, that animals treated with peroxide of hydrogen after infection succumbed to diphtheria much quicker than the control animals; and as I added this substance to the diphtheria cultures in quantities approaching the fatal dose, I found the virulence of such cultures considerably heightened. On the other hand, when I some days later infected animals previously treated with peroxide of hydrogen, they had acquired a pronounced degree C?f immunity, which in guinea-pigs was shown, among other things, by the fact that there was not observable at the seat of infection very diffuse <:edema spreading to the surrounding parts, but a firm tumour tending to suppuration. On cutting into this there flowed out a clear serous fluid, which was enclosed by a dense envelope. The experiments with peroxide of hydrogen are still being pursued, and I cite here only one series of them, which, according to a common project, have been carried out in this institute by Herr Stabsarzt Ltibbert. Each of five rabbits received on the 11th, 12th, 14th, and 17th November 5 ccm. of peroxide of hydrogen. On the 20th November, in presence of Ltibbert, I injected subcutaneously '5 ccm. of perrectly virulent diphtheria culture in bouilloll to these five animals, and at the same time to a fresh rabbit. The control animal died of typical diphtheria on the 21st November, that is, after twenty-four hours. Of the previously treated animals one died on the 25th November (after five days), two on the 27th November (after seven days), one on the 28th November (after eight days), and one showed no symptom of illness and has remained healthy. Of the five different modes of immunity against diphtheria previously mentioned, the last four belong to those which Pasteur first taught us. The fifth, which brings about immunity by substa,nces quite foreign to the agents that excite the infection-indeed, of a perfectly simple chemical nature. has as yet scarcely any analogy. That the possibility of producing immunity by simple chemical substances exists also in the case of other infectious diseases can be proved by the' fact which. through agreement with Herr Kitasato, I here report, namely, "that one of the metheds of rendering rabbits immune against tetanus consists in previously treating them exclusively with trichloride of iodine solution." The whole five of the already-described methods' of producing immunity against diphtheria are, at least in the form in which I here report them, in my opinion not applicable to the human subject. But from a scientific point of view, and especially for the solution of
GENERAL ARTICLES.
the problem with which alone I am here concerned-for the comprehension of the production of immunity against diphtheria, they are capable of rendering valuable service. However the immunity may be brought about, and I do not exclude the natural immunity against diphtheria, alI diphtheria-immune animals have in common certain properties which distinguish them from non-immune animals. In the first place, alI living immune animals are protected, not only against infection with the living baciIIi, but also against the deleterious action of those poisonous substances which are formed in cultures and in the animal body by diphtheria bacilli. The investigation of this I have carried out in different ways. In the first place, I experimented with the solution of an albuminous substance which I separated from old cultures by means of acid alcohol; I was, however, unable to remove the acid from the preparation obtained without injury to the action of the poison. I also found it no easy task to separate from the precipitate ol::itained other accessory substances, although the earlier investigators in this sphere succeeded without difficulty. But for the present purpose it was not necessary to separate the diphtheria poison-ot, perhaps it would be more correct to say, the diphtheria poisons., Filtered old cultures afforded me alI that I wanted. I found that after ten weeks my cultures grown in alkaline bouilloll with 10 ccm. normal alkaline solution per litre, contained so much poisonous substance that, when made germ-free by filtration, a dose of I ccm. produced in medium-sized guinea-pigs characteristic symptoms of diphtheria poisoning, which entirely disappeared only after 3-4 weeks. From 3-4 ccm. suffice to kill even larger guinea-pigs after 3-8 days. Necrosis of the skin occurred very regularly, and, indeed, not simply at the seat of infection, but quite remote from thatmost frequently on the belly. AlI the guinea-pigs which now possessed a firm diphtheria immunity-that is such as no longer showed any symptoms of illness even after repeated infection-bore 3-5 ccm. without exhibiting any symptoms of poisoning or any local reaction whatever. On the other hand, the guinea-pigs not quite recovered from an infection showed' themselves only very little more resistant to the poison than normal animals, and necrosis of the skin appeared in the same manner as in the latter. It is very remarkable that the immunity can again be lost in consequence of the subcutaneous injection of large and repeated doses of the poison; this occurs the more certainly, the less firmly fixed the immunity has been. In every case the guinea-pigs under the influence of the poisonous germ-free diphtheria culture are in less favourable conditions towards diphtheria infection than before. One might at first suppose that the resistance to poison here described depends upon a "poison habit," such as is alIeged to exist in the case of drunkards, opium eaters, and arsenic eaters, certain vital centres ceasing to react to the particular poison-in short that it is in reality a case of accustoming or inuring of the vital organs. But such a view is here immediately opposed to the fact that even such animals as never had anything to do with the diphtheria poison possess the power of resistance to that poison. To return again to the germ-free ten-weeks-old culture, that is,
GENERAL ARTICLES.
41
reckoned by body-weight, fatal to guinea-pigs in the proportion of about I: roo; but mice stand the poison without injury when it is injected into them on the proportion of I : 20; and I have injected into rats 4 ccm. on several successive days without setting up any reaction worth naming. Opposed to the view of a poison habit there is the further fact that I could not succeed by the most careful ascent from quite innocuous to higher doses of the poison in protecting the animals against the diphtheria poison so as t<;> make them ,subsequently tolerate a little more of it than normal animals. These observations and reflections led me to consider more closely whether the cause of the power of resistance to the poison may depend, not on a peculiarity of the living cellular parts of the organism, but upon a special peculiarity of the blood, apart from living cells. In order to decide this question, I took blood from rats into the peritoneal cavity of which large quantities of the diphtheria poison had been introduced three hours previously, and injected this blood or the serum obtained from it into the peritoneal cavity of guinea-pigs; no symptoms of poisoning appeared in the latter, whereas the blood of animals susceptibJe to diphtheria, and which had received the diphtheria poison in the same quantity into the peritoneal cavity, while it did not kill guinea-pigs, at least made them distinctly ill. Further, I attacl;1 importance to the fact that even the extravascular blood of guinea-pigs immune against diphtheria possesses the power to render the diphtheria poison innocuous. To what degree this is the case, and to what extent therapeutic results may be obtained with the blood of animals rendered immune, I intend to consider in a later communication. I have still to mention a negative criterion common to immune animals. Before I had arrived at those investigations which enabled me to find recognisable distinctions between the blood of animals susceptible to diphtheria and that of those immune against the disease, I had also employed the methods hitherto in use in examining blood. I had in this way examined the blood of four guinea-pigs and two rabbits that had been rendered immune, as well as the blood of over ten rats, as to whether there could be recognised in it any influence adverse to bacteria. Such was not the case; in all cases the diphtheria bacilli grew very luxuriantly, and their virulence was rather increased. After I had made these observations in my investigations concerning the production .of diphtheria immunity, Herr Kitasato and I in common applied them to the case of tetan.us; and in our first communication we have succeeded, as we think, in furnishing the indisputable proof that in tetanus the poison-destroying action of the blood of tetanus-immune animals is a causa sujjiciens for the production of the immunity. The fortunate circumstance that we could obtain from an immune rabbit so much blood and serum with which to treat a larger number of the much smaller mice, has made it possible to expose the therapeutic consequences fully and completely. It is possible that in my article published in common with Kitasato the curative effect of the transfusion of blood from tetanus-immune rabbits was not expressed with sufficient distinctness, and I will therefore here give special
GENERAL ARTICLES.
prominence to the statement that mice are not merely rendered imll?-uQe, I;>y the blood of tetanus-immune rabbits, and that the protection of mice is brought about not m(C:rely when blood or serum ,of immune animals is injected into their peritoneal cavity immediately after infection, and before they have contracted tetanus; even }vhen severa! extremities have already becom~ tetanic, and when, accorqing, to previous experience, they might be expected to die in a few, hours if not treated-even then a cure can be effected with great c,ert!linty, and, indeed, so rapidly that within a few days no trace of disease is observable. The,possibility of a cure in the case of diseases with a quite acute course can therefore no longer be denied.
ON EXTRA-UTERINE PREGNANCY IN THE CAT. By D.
J.
HAMILTON, M.B., Professor of Pathology,
Aberdeen University.
QUITE apart from the practical issues bound up with it, the subject of extra-uterine pregnancy considered from the purely biological point of view is one of great interest. The case which I am about to describe occurred in a cat, and is worth recording from extra-uterine pregnancy being a somewhat rare condition in the lower animals, as well as from the unique attachments which the progeny had contractedwith neighbouring parts. It is asser,ted that there are three situations in which primarily the embryo or embryos may be found; Firstly, occupying a Fallopian tube; secondly, within the ovary itself; and, t/lirdly, growing from the peritoneum. The great majority of extra-uterine pregnancies, in Man at least, are acknowledged on all hands to be tubal in their origin. The fcetus grows within the tube until about the end of the second or the beginning of the third month. It then bursts forth in one of two directions, either into the peritoneum or into the loose cellular tissue between the layers of the broad ligament. In the case of the fcetus escaping into the peritoneal sac, which is the commoner of the two occurrences, the parent dies sooner or later if unrelieved, and usually from abdominal ha:-morrhage. The fcetus, however, on rupturing into the abdomen need not im1llediate!.y perish, nor need the condition prove at once fatal to the mother. On the contrary, the embryo may continue to grow in the peritoneal cavity until full time is reached, the placenta remaining adherent to the Fallopian tube, and the umbilical cord escaping through the rupture in its wall. The lacerated edges of the wound cicatrise, so that if examined some time after the accident has happened the cord is found to issue through an aperture with a regular clean cut margin (Sa Chiari's case, Zcitscltrift fltr Heilkunde, vol. viii., 1887, p. 127). Although the tube usually ruptures, yet it has been alleged that in some instances it goes on stretching up to the full term of gestation. A case recorded by Chiari (loc. cit., p. 128) appeared to have been of this nature. The child was fully nourished and was found lying in a sac supposed to be the distended Fallopian tube. A layer of muscular fibre lined the sac, said to be that of the tube in a state of