v^n THE LIBRARY OF THE UNIVERSITY OF CALIFORNIA LOS ANGELES GIFT OF SAN FRANCISCO COUNTY MEDICAL SOCIETY ^ ^-^^Ui-^r^ u ^ ^^Amjjuuvotoplasin of the cajjfnred bacterium, thus putthig an end to the possibility of its doing any harm. " In the case of a pathogenic organism we may imag- ine that, when captured in this way, it may share a like fate if the captor is not paralyzed by some potent poison evolved by it, or overwhelmed by its superior vigor and rapid multiplication. In the latter event the active ca- reer of our conservative white corpuscles would be quickly terminated and its protoplasm would serve as food for the enemy. It is evident that in a contest of this kind the balance of power would depend upon circumstances relating to the inherited vital characteristics of the invad- ing parasite and of the invaded leucocyte." 16 IMMUNITY AND SERUM-THERAPY. This explanation is now very commonly spoken of as the " Metschuikoff tlieoiy," althoiig-h, as a matter of fact, it was clearly stated by the writer several years (1881) before Metschuikofl"s first j^aper (1884) was published. Metschnikoff has, however, been the principal defender of this explanation of acquired immunity, and has made extensive and painstaking researches, as a result of which many facts have been brought to light which ajD- l^ear to give support to the present writer's hypothesis — the so-called Metschnikoff theor\\ The observations which first led Metschuikoff to adopt the explanation of immunity under consideration were made u^Don a species of daphuia, which is subject to in- fection by a torula resembling the yeast fungus. Enter- ing with the food, this fungus penetrates the walls of the intestine and invades the tissues. In certain cases the infection does not prove fatal, owing, as Metschnikoff as- serts, to the fact that the fungus cells are seized upon by the leucocytes, which appear to accumulate around the invading parasite (chemiotaxis) for this special purpose. If they are successful in overpowering and destroying the parasite, the animal recovers ; if not, it succumbs to the general infection which results. In a similar man- ner, Metschnikoff' supposes, pathogenic bacteria are de- stroyed when introduced into the body of an immune animal. The colorless blood-corpuscles which he de- nominates j)hagocytes, accumulate at the point of inva- sion and pick up the bacteria, as they are kno^Ti to pick up inorganic particles injected into the circulation. So far there can be no doubt that Metschnikoff' is right. The presence of bacteria in the leucocytes in considera- ble numbers, both at the point of inoculation and in the general circulation, has been repeatedly demonstrated NATUEAL IMMUNITY. 17 in animals inoculated with various pathogenic bacteria. The writer observed this in his experiments, made in 1881, in which rabbits were inoculated with cultures of his " Micrococcus Pasteuri " (micrococcus of croupous pneumonia), and it was this observation which led him to suggest the hypothesis which has since been so vig- orously supported by Metschnikoff. But the presence of a certain number of bacteria within the leucocytes does not prove the destructive power of these cells for living pathogenic organisms. As urged by Weigert, Baumgar- ten, and others, it may be that the bacteria were already dead when they were picked up, having been destroyed by some agency outside the blood-cells ; and, as we shall see later, there is experimental evidence that blood- serum has decided germicidal power for certain patho- genic bacteria, and that the blood-serum of the rat and other animals, which have a natural immunity against anthrax, is especially fatal to the anthrax bacillus. On the other hand, there is reason to believe that living bac- teria picked up by leucocytes are not always destroyed, but that, in some instances at least, they thrive and mul- tiply within these protoplasmic masses. In mouse sep- ticaemia and in gonorrhoea one would be disposed to decide, from the appearance and arrangement of the pa- thogenic bacteria in the leucocytes, that tliey are not de- stroyed but that they multiply in the interior of these cells, which in the end succumb to this parasitic inva- sion. In both of the diseases mentioned we find the leu- cocytes so completely filled with the pathogenic micro- organisms that it is difficult to believe that they have all been picked up by a voracious phagocyte, which has stuffed itself to repletion, while numerous other leuco- cj'^tes in the same microscopic field have failed to capture 18 IMMUNITY AND SERUM-THEIIA 1>Y. a single bacillus or micrococcus. Moreover, the staining of the parasitic invaders, and the characteristic arrange- ment of the " gonococcus " in stained preparations of gonorrhoeal pus, indicate that their vitality has not been destroyed in the interior of the leucocytes or pus-cells, and we can scarcely doubt that the large number found in cei-tain cells is due to multiplication /// sita rather than to an unusual activity of these particular cells. But in certain infectious diseases, and especially in inoculations of the anthrax bacillus into immune animals, the bacilli included within the leucocytes often give evidence of de- generative changes, which would support the view that they are destroyed within the leucocytes, unless these changes occurred before they were picked up, as claimed by Nuttall and others. Metschnikoif divides the so-called phagocytes into two grouj)s : Fixed phagocytes (endothelial cells, etc.) and free phagocytes. According to his observations not all leucocytes are phagocytes. The comparatively small, immobile leucocytes (" lymphocytes ") with a single large nucleus never take up bacteria. The large uninu- clear leucocyte which exhibits active amoeboid move- ments, and in which the nucleus is frequently lobed or reniform, is called by Metschnikoff a macrophage, and the smaller multinuclear leucocyte (or with one nucleus in process of breaking up) he calls a microphage. The macrophages are believed to be largely of endothelial origin. Metschnikoff asserts that in general the more virulent the microorganism the rarer is its presence ob- served within the iDhagocytes. Thus in the acute sep- ticaemias which are quickly fatal to siisceptible animals — e.g., fowl cholera, rabbit septicaemia, anthrax in mice, etc. — the pathogenic bacteria are rarely found in the inte- NATURAL IMMriNITY, 19 rior of the cells. They remain free in the vicinity of the point of inoculation and thence quickly invade the blood. On the other hand, in infectious diseases having- a more protracted course there is a very decided phago- cytosis. As an example of this Metschnikoff mentions mouse septicaemia " which has a duration in the mouse two and a half times as long- as that of anthrax in the same animal," and the chronic infectious diseases — tuber- culosis, leprosy, rhinoscleroma, and g-landers. Ag-ain, at- tention is called to the phenomena attending recovery from certain infectious diseases. In relapsing- fever, for example, during the sudden access of the fever the spi- rilla are present in the blood in great numbers, and they are not included within the cells. During the apyretic stage they disappear from the blood, but are found in- cluded in the phagocytes of the spleen. In like manner rats and pigeons which survive an attack of anthrax, when killed during the period of resolution show numer- ous bacilli included within leucocytes and splenic phago- cytes, and very few free bacilli. In a similar manner, according to Metschnikoff, ani- mals which have an acquired immunity for anthrax or other infectious diseases of bacterial origin, when inocu- lated subcutaneously, resist infection because there is an extensive emigration of leucocytes to the jDoint of inocu- lation, and these soon include the bacteria in large num- bers. In susceptible animals, not having thus acquired immunity, the phagocytes are inoperative, and the bac- teria are very rarely seen included within them. As ex- amples of this Metschnikoff cites the bacillus of anthrax and Vibrio Metschnikovi. In unvaccinated rabbits in- oculated with anthrax, phagocytosis is said to be very seldom seen, while in rabbits protected by " vaccination " 20 IMMUNITY AND SERUM-THKKAPY. it is veiy marked. The differance is still more remarka- ble in guiDea-pigs inoculated with Vibrio Metschnikovi. In vaccinated animals the phagocytes are loaded with the microbes, in those not vaccinated they are not seen included in these cells. According to Metschnikoff the giant cells of tuberculosis are " huge multinuclear phago- cj'tes " in which the bacilli are destroyed. This is shown by the " very evident signs of degeneration ; the bacilli swell, their enveloping membrane becomes much thick- ened and highly refractive, and in time the contents lose the power of fixing the staining material, so that, event- ually, nothing is left but slightl}^ yellowish forms, re- calling, in propoiiions and position, the enlarged ba- cilli." Actio)i of Blood-Serum and OtJier Organic Liquids Upon Bacteria. — Bacteriologists have long been aware of the fact that many species of bacteria, when injected into the circulation of a living animal, soon disappear from the blood, and that the blood of such an animal a few hours after the intravenous injection, of putrefactive bacteria for example, does not contain living bacteria capable of development in a suitable culture medium. Wyssoko- witsch, in an extended series of experiments, has shown that non -pathogenic bacteria may be obtained in cultures from the liver, spleen, kidneys, and bone marrow after they have disappeared from the blood ; but that, as a rule, those x^resent in these organs have lost their vital- it}^ as shown by culture experiments, in a period varying from a few hours to two or three days. As has been stated above, this disappearance was accounted for by the writer (1881), and later by Metschnikoff (1884), as the result of the vital activities of the leucocytes ; but more recently experimental evidence has been presented which NATURAL IMMUNITY. 21 indicates that tliis is not the only, and probably not the principal, agency by which bacteria introduced into the circulation of living animals are destroyed. Von Fodor (1887) first called attention to the fact that anthrax bacilli may be destroyed by freshly drawn blood ; and Nuttall (1888), in an extended series of experiments, showed that various bacteria are destroyed within a short time by the fresh blood of warm-blooded animals. Thus the anthrax bacillus in rabbit's blood was usually killed in from two to four hours when the temperature Avas maintained at 37° to 38° C, and the same result was obtained with pigeon's blood at 41° C. But when the blood was allowed to stand for some time, or was sub- jected to a temperature of 55° C, it no longer joossessed germicidal properties and served as a culture-fluid in which an abundant development of anthrax bacilli oc- curred. Bacillus subtilis and Bacillus megatherium were also destroyed by fresh rabbit's blood, but it was without action upon Staphylococcus pj^ogenes aureus, which, at a temperature of 37.5° C, was found to have increased in numbers at the end of two hours. Further researches by Behring and Nissen (1890) show that there is a wide dif- ference in the blood of different animals as to germicidal power, and that certain bacteria are promptly destroyed, while other species are simply restrained for a time in their development or are not affected. Thus Nissen found that the cholera spirillum, the bacillus of anthrax, the bacillus of typhoid fever, and Friedliinder's bacillus were killed, while Staphylococcus pyogenes albus and aureus. Streptococcus pyogenes, the bacillus of foAvl cholera (Bacillus septicaemiae haemorrhagicge), the bacil- lus of rothlauf and Proteus hominis were able to mul- tiply in rabbit's blood after having been restrained for 22 IMMUNITY AND SERUM-TIIEUAPY. a short time in their development. In the case of the cholera spirillum a period of ten to forty minutes suf- ficed for the complete destruction of a limited number, but when the number exceeded 1,200,000 per c.c. they were no longer destroyed with certaint}^ and after five hours an increase occurred. The anthrax bacillus was commonly destroyed within twentj^ minutes, and the typhoid bacillus at the end of two hours. In the experi- ments of Behring- and Nissen it was found that the most pronounced germicidal eflect was obtained from the blood of the rat, an animal which has a natural immunity ag-ainst anthrax ; while the blood of the g-uinea-pig-, a verj^ susceptible animal, had no restraining effect and served as a favorable culture-medium for the anthrax ba- cillus. And the remarkable fact was developed that when the blood of the rat was added to the blood of the guinea- XJig", in the proportion of 1 to 8, it exercised a decided restraining- influence on the growth of the anthrax bacil- lus. Later researches have shown that cultivation in the blood of an immune animal causes an attenuation of the virulence of an anthrax culture (Ogata and Jasuhara) ; also that the injection of the blood of a frog or of a rat into a susceptible animal which has been inoculated with a virulent culture of the anthrax bacillus will restrain the development of the pathogenic bacillus and prevent the death of the inoculated animal. Buchner (1889) first j)roved by experiment that the germicidal power of the blood of dogs and rabbits does not depend upon the jiresence of the cellular elements, but is present in clear serum which has been allowed to separate from the clot in a cool place. Exposure for an hour to a temperature of 55° C. destroys the germicidal action of serum as well as of blood ; the same efiect is NATURAL IMMUNITY. 23 produced by heating to 52° C. for six hours, or to 45.G'^ C. for twenty hours. The germicidal power of blood-serum is not destro^^ed by freezing- and thawing, but is lost after it has been kept for some time at ordinary tempera- tures. The researches of Buchner, of Hankin, and others, show that this germicidal power of fresh blood-serum depends upon the presence of proteids, to which the first-named bacteriologist has given the name of " alexins." Hankin, in his paper upon the origin of these " defensive pro- teids " in the animal body (1892), arrives at the conclusion that while they are present in the cell-free serum they are the product of certain leucocytes — Ehrlich's eosino- phil cells. He believes that the eosinophil granules become dissolved in the serum and constitute the germi- cidal proteid which is shown to be present by experi- ments upon bacteria. According to Hankin the separ- ation of these granules can be witnessed under the microscope. They first accumulate upon one side of the cell and then gradually disappear, and as this occurs a considerable increase in the bactericidal power of the serum can lie demonstrated. The germicidal power of the blood-serum is also said to be increased when the number of leucocytes is considerably augmented, as oc- curs when a sterilized culture of Vibrio Metschnikovi is injected subcutaneously. Also by treatment which favors a separation of the alexin from the leucocytes, i.e., a so- lution of the eosinophil granules. This may be accom- plished by the injection of an extract of the thymus gland of the calf, or by simply alloAving the drawn blood to stand for several hours at a temperature of 38'' to 40" C. Buchner's latest communication upon the subject shows that he also attributes the origin of the germicidal 24 IMMUNITY AND SEItUM-TIIEILVPY. proteid in fresh blood-serum to the leucocytes. In his paper on " Immunity," read at the Eighth International Congress on Hygiene and Demography (Budapest, 1894) he calls attention in the first place to the fact that a clearly marked distinction must be made between natu- ral immunity and acquired immunity, inasmuch as the " alexins " and " antitoxins " have "very different prop- erties. The first-mentioned proteids are destroyed by a comparatively low temperature (55° to 60° C.) while the antitoxins resist a considerably higher temperature, and, unlike the alexins, have no bactericidal or globulicidal action. A very remarkable fact developed in Buchner's experiments is that the blood- serum from the dog and from the rabbit, when mixed, neutralize each other so far as their germicidal power is concerned. By injecting sterilized emulsions of wheat-flour paste in- the pleural cavity of rabbits and dogs Buchner suc- ceeded in obtaining an exudate which had more decided germicidal power than the blood or serum of the same animal. This was evidently due to the large number of leucocytes present, but not to their phagocytic action, as was shown by experiment. By freezing the exudate the leucocytes were killed, but the germicidal action of the fluid was rather increased than diminished by freezing. While freezing had no efi"ect upon the germicidal action of the pleural exudate this was always neutralized by exposure to a temperature of 55° C. The observations referred to lead Buchner to the conclusion, which we con- cur in, that phagocytosis plays an entirely subordinate role in the germicidal action of freshly drawn blood, but that this action does depend, to a considerable extent at least, vxpon the leucocytes, inasmuch as the soluble pro- teid to which it is due has its origin from these proto- NATURAL IMMUNITY. 25 plasmic masses. Bucliner says in his recent paper, above referred to : " Upon the basis of tbe results readied a reconciliation with the theory of phagocytosis seems very possible, as the researches upon the bactericidal action of blood and serum lead to the final conclusion that the leucocytes are the bearers of the bactericidal material. The extended facts of observation which Metschnikoff and his associ- ates have collected in favor of phagocytic action remain undisturbed. But the explanation of the process, the conception of the causal connection, must be somewhat different. Through sucli a conception the observations opposed to the theory of phagocytosis will also find their explanation. Tlius in the observations of Eibbert, which led to the statement of his wall-forming theory, the ' mantel ' of leucocytes which surrounds the infected focus may very well represent a protecting wall which acts, not by phagocytosis but through the germicidal proteid given off by the leucocytes. Also in streptococ- cus infection recovery often occurs without any evidence of phagocytosis. Miiller, in recent experiments on an- thrax in rats, has failed entirely to observe phagocytosis, although the organism of the rat is very unfavorable for anthrax infection. Also in relapsing fever Tictin was not able to discover any phagocytosis in apea from which the spleen had been removed, either during the attack or after recovery, when this occurred. And E. Pfeiffer re- cently demonstrated, in his interesting researches upon the killing of cholera vibrios in the peritoneal cavity of immunized guinea-pigs, that their rapid destruction oc- curred without phagocytosis. All of these cases present no difficulty as soon as the idea is abandoned that the act of devouring (' Auffressen ') constitutes a conditio sine qua non for the bactericidal activity of the leucocytes." Emmerich, Tsuboi, Steinmetz, and Low (1892), as a re- sult of extended experiments, arrived at the conclusion that the germicidal action of blood-serum " depends 26 IMMUNITY AND SERUM-THERAPY. upon a specific propei*ty of the alkali-serum-albumin, and that it is a purely chemical process." The}^ state that when the germicidal power is neutralized by heat it may be restored by the addition of an alkali. Buchner repeated the experiments of Emmerich and his associates and obtained similar results, but interprets them differ- ently. According" to him the serum does not regain its germicidal power, but after the addition of an alkali and subsequent dialyzing the nutritive value of the serum is so diminished that the bacteria do not develop in it. The failure of the anthrax bacillus to develop in inocu- lated white rats has been ascribed by Behriug (1888) to the highly alkaline reaction of the blood and tissue juices of this animal. Hankin (1891) in extended experiments arrived at a different conclusion. From the spleen and blood-serum of rats he isolated a globulin possessing germicidal properties, to which he ascribes the power of the rat's blood to destroy anthrax bacilli, without, how- ever, rejecting the view that the excessive alkalinity of the blood of this animal may be a factor in producing this result. Pane (1892) has made experiments which give additional weight to the assumption that the alka- linity of the blood is an important factor in accounting for immunity. He states that carbonate of soda, dissolved in water, in the proportion of 1 to 3,000, has a decided germicidal action upon the anthrax bacillus, equal to that of the blood-serum of the rabbit. And that when rabbit serum is completely neutralized it no longer has any in- jurious action on anthrax bacilli. Zagari and Innocente (1892) also arrived at the conclu- sion that the diminished resistance to anthrax infection resulting from curare-poisoning in frogs, and from chloral or alcohol in dogs (Platania), in fowls as a result NATURAL IM3IUNITY. 27 of starvation (Canalis and Morpurgo), in white mice as a result of fatigue (Cliarin and Roger), is, in fact, due to di- minished alkalinit}^ of the blood, which they found to cor- respond wdtli the increased susceptibility resulting from the causes mentioned. Buchner (1892) states that several of the ammonium salts, and es^Decially ammonium sulphate, cause an in- crease in the germicidal action of blood-serum, and also increase its resistance to the neutralizing effects of heat. The experiments of Pansini and Calabrese (1894) show, on the contrary, that the addition of uric acid to blood- serum diminishes its bactericidal activity, as does also the presence of glucose. That certain infectious dis- eases are especially virulent in persons suffering from diabetes is a frequentlj" repeated clinical observation. Van Fodor has shown by experiment that the injection of an alkali into the circulation of a rabbit increases its resistance to anthrax infection and the germicidal activ- ity of its blood-serum. The same bacteriologist has found that when a rabbit is infected with anthrax, the al- kalinity of its blood is notably increased during the first twenty-four hours, when we may suppose that the pow- ers of nature are brought to bear to resist the invading parasite, and that after this time it rapidly diminishes. Ten hours after infection (by subcutaneous inoculation?) the alkalinity of the blood had increased 21.5 per cent. Shortly before the death of the animal a diminution of 26.3 per cent, was noted. This diminution w^as observed in thirty-four out of thirty-nine animals experimented upon, and these animals succumbed to the anthrax infec- tion in a shorter time than did the other five in which there was no such diminution. It seems probable that the germicidal property of 28 IMMUNITY AND SERUM-THEKAPY. freshly drawn blood-serum is not due to its alkalinity, per sc, but to the fact that the germicidal constituent is only soluble in an alkaline fluid. The recent researches of Vaug-hn, McClintock, and Nov}^ indicate that this germicidal constituent is a nuclein. Dr. Vaughn in his last iDublished paper upon " Nucleins and Nuclein The- rapy," says : " Kossel, of Berlin, has confirmed our state- ments concerning the germicidal action of the nucleins. Dr. McClintock and I have also demonstrated that the germicidal constituent of blood-serum is a nuclein. This nuclein is undoubtedly furnished by the pol^^nu- clear white corpuscles." Denys has recently (1894) re- ported the results of experiments made in his laboratory by Yan der Velde, which give support to the conclusion reached by Vaughn. In these experiments a sterilized culture of staphylococci was injected into the pleural cavity of rabbits in order to obtain an exudate. At in- tervals of two hours this exudate was obtained by killing one of the animals in the series experimented upon, and at the same time blood from the animal was secured. Both the exudate and the blood was placed in a centrifu- gal machine, in order to obtain a serum free from corpus- cular elements. The germicidal activity of the serum was then tested. The general result of the experiments was to show that the longer the interval after the injec- tion into the pleural cavity, the more potent the germi- cidal activity of the exudate became ; and that there was no corresponding increase in the activity of tlie blood- serum obtained from the circulation. At the end of ten or twelve hours, the serum from the exudate killed all of the staphylococci in a bouillon culture twenty times as great in quantity as the germicidal serum used in the ex- periment. The absence of any increase in germicidal NATURAL IMMUNITY. 29 power iu tlie blood-serum taken from the general circu- lation shows that the notable increase manifested by the exudate was due to local causes ; and as a matter of fact it corresponded with an increase in the number of leuco- cytes as found in the pleural exudate. Thus it will be seen that the independent researches of Haukin, of Buchner, of Vaughn, and of other competent bacteriologists, have led them to the same ultimate re- sult so far as the origin of the germicidal constituent of the blood is concerned, and that the leucocytes aj)pear to play an important role in the protection of the animal body from invasion by bacteria (natural immunity), al- though the method by which this is accomplished differs from that suggested by the writer in 1881, and since strongly supported by Metschnikofi' and his associates — " phagocytosis." With reference to the physiological and chenjical char- acters of the nucleins I quote from Vaughn's paper, above referred to, as follows : " Phj^siologically nucleins may be said to form the chief chemical constituent, of the living parts of cells. Speaking broadly, we may say that the nuclein is that constituent of the cell by virtue of which this histologic unit grows, develops, and reproduces itself. It is the function of the nuclein of the cell to utilize the pabidum within its reach. It must be evident that those tissues most abounding in celhdar elements contain relatively the largest amounts of nuclein. It must also be seen that it is by virtue of their nuclein that the cells of "various organs and organisms possess and manifest their indi- vidual peculiarities. We should therefore expect to find that the nuclein of the yeast-cell is not identical with that of the bacillus tuberculosis, and that the nuclein of the spleen differs from that of the thj^roid gland. The number of kinds of nuclein is limited only by the varie- 30 IMMUNITY AND SERU^I-TlIEKAPy. ties of cells. Nuclein is the chemical basis of that part of the cell designatecl by the histologist as the nucleus, sometimes called chromatin on account of the readiness with which it absorbs and holds coloring agents. It is the nuclein of the bacterium which takes up and retains the stains, and it is on account of the fact that the nuclein of the bacillus tuberculosis differs from that of other bacilli that we are able to distinguish the former from the latter by its tinctorial properties. Differences in re- action with staining reagents, so plainly seen under the microscope, are only outward manifestations of less ap- jiareut and more important differences in chemical com- position. " Chemically the nucleins are complex, proteid bodies, characterized especially by the large amount of phos- phorus which they contain. The phosphorus exists in the form of nucleiuic acid, which is combined with a highly complex basic substance. So far as we know at present, the nucleinic acid of all nucleins is the same, yet the basic part differs in the various nucleins. This basic substance yields, as decomposition products, one or more of the so-called xanthin bodies ; adeuin, guanin, sarkin, and xanthin. Some nucleins yield only adenin, and these may be designated as adenyl nucleinic acids. Those which furnish xanthin most abundantly may be called xanthyl nucleinic acids. Generally ^pQaking, the nucleins are insohible in dilute acids and soluble in dilute alkalies. They resist peptic digestion and in this way maj^ be se]i- arated from most other proteid bodies." It has been shown by several investigators that the number of leucocytes increases in certain infectious dis- eases, and this increase, together with an increased alka- linity of the blood, which has heretofore been referred to, appears to be a provision of nature for overcoming the infection which has already occurred. Billings,* in a re- * J. S. Billings, Jr. : The Leucocytes in Croupous Pneumonia, Bulletin of the Johns Hopkins Hospital, November, 1894, p. 105. NATURAL IMMUNITY. 31 cent paper, gives the evidence as regards croupous pneu- monia. Some of his conclusions are given below : " In cases of pneumonia pursuing a favorable course there is, as a rule, a marked increase in the number of leucocytes during the febrile iDeriod of the disease. This leucocytosis is probably present at the time of the chill, and may be very marked within a few hours." " In cases showing extensive involvement of both lungs the leucocytes are apt tp reach a higher point than in those cases where the involvement is only moderate." " The fatal cases may show either the ijresence or ab- sence of leucocytosis. In those cases showing a leucocy- tosis, some other cause of death than the virulence of the bacterial poison must be sought for." " The prognosis in cases sliowing a complete and con- tinuous absence of leucocytosis is unfavorable as a rule." " The leucocytosis in pneumonia is a so-called pure leucocytosis, i.e., an increase in the poly nuclear elements solely." The experimental evidence submitted, considered in connection with the extensive literature relating to " pha- gocytosis," leads us to the conclusion that natural immu- nity is due to a germicidal substance present in the blood- serum which has its origin (chiefly, at least) in the leu- cocytes and is soluble only in an alkaline medium. And that local infection is usually resisted by an afilux of leu- cocytes to the point of invasion ; but that phagocytosis is a factor of secoudary importance in resisting parasitic invasion. Also that general infection, at least in some infectious diseases, is resisted, and in non-fatal cases over- come, by an increase in the number of leucocytes and in the alkalinit^^ of the blood-serum — which favors solution of the germicidal proteids contained in the polynuclear leucocytes. 32 IMMUNITY AND SEUUM-TIIEHAPY. This conclusion is in accord with certain other facts which may be briefly referred to. Numerous experi- ments show that natural immunity may be overcome by infection with an excessive number of pathogenic bacte- ria and their products, as contained in a culture ; or by an unusually virulent variety. According to Denys the pathogenic bacteria, in general, resist the germicidal ac- tion of rabbit serum better than the saprophytic bacte- ria ; and of two varieties of the same pathogenic species the most virulent has the greater resistance to this ger- micidal action. This is in accord witli the fact that nat- ural immunity protects completely from invasion by the ordinary saprophytes, by which man and the lower ani- mals are surrounded on all sides. It is only when the circulation is arrested in the entire body, or in a portion of it, that the putrefactive bacteria succeed in invading the tissues. The action of the germicidal substance present in blood-serum upon bacteria appears to be a quantitive one so far as the bacteria are concerned, and it may also be neutralized by their products. Thus Bastin (1892) concludes from his experiments that the germicidal ac- tion of blood -serum from the dog is neutralized or greatly diminished by the intravenous injection of cult- ures of bacteria (S. pyogenes aureus, Bac. lactis ?ero- genes) in considerable amounts, and that this occurs as well when the bacteria in the cultures have been killed l)y heat as when they are living. This effect is mani- fested Avithin two minutes after the injection, and is said to reach its maximum inside of ten minutes ; at the same time there is a diminution in the coagulability of the blood. At the end of five or six hours the normal coagu- lability and germicidal action has been recovered. The NATURAL IMMUNITY. 33 same is true as regards freshly drawn blood, hi vitro. When bacteria or their products are added in excess the germicidal action is no longer manifested, but multi- plication occurs at once, as in a favorable culture-fluid. Thus Nissen found that a limited number of cholera spirilla added to freshly drawn rabbit's blood perished in from ten to forty minutes ; but when the number ex- ceeded 1,200,000 per cubic centimetre an increase oc- curred at the end of five hours. It has been demonstrated by several experimenters that other albuminous fluids possess a germicidal power similar to that manifested by freshly drawn blood. Thus Nuttall found that a pleuritic exudate from man de- stroyed the anthrax bacillus in an hour, the aqueous humor of a rabbit in two hours. Prudden found that the albuminous fluid obtained from a hydrocele sac, or from the abdominal cavity in ascites, possesses similar germi- cidal power, and Fokker has demonstrated that fresh milk destroys the vitality of certain bacteria. Hankin has extracted from the spleen and lymphatic ylands of dogs and cats a " cell' globulin " which pos- sesses germicidal power similar to that of blood-serum. This he obtained by treatment with a solution of sodi- um chloride and subsequent precipitation with alcohol. This " globulin " is insoluble in water or alcohol, and does not dialyze. n. ACQUIRED IMMUNITY. It has long been known that, in a considerable number of infectious diseases, a single attack, however mild, af- fords protection against subsequent attacks of the same disease ; that in some cases this protection appears to be permanent, lasting during the life of the individual ; that in others it is more or less temporary, as shown by the occurrence of a subsequent attack. The protection afforded by a single attack not only differs in different diseases, but in the same disease varies greatly in different individuals. Thus certain individu- als have been known to suffer several attacks of small-pox or of scarlet fever, although, as a rule, a single attack is protective. Exceptional susceptibility or insusceptibil- ity may be not only an individual but a family character- istic, or it may belong to a particular race. In those diseases in which second attacks are not in- frequent, as, for exami^le, in i)neumonia, in influenza, or in Asiatic cholera, it is difficult to judge from clinical ex- perience whether a first attack exerts any protective in- fluence. But from experiments upon the lower animals we are led to believe that a certain degree of immunity, lasting for a longer or shorter time, is afforded by an attack of pneumonia or of cholera, and probably of all infectious diseases due to bacterial parasites. In the malarial fevers, which are due to a parasite of a different ACQUIRED IMMUNITY. 36 class, one attack affords no protection, but rather j)redis- poses to a subsequent attack. In those diseases in which a single attack is generally recognized as being protective exceptional cases occur in which subsequent attacks are developed as a result of unusual susceptibility or exposure under circumstances especially favorable to infection. Maiselis has recently (1894) gone through the literature accessible to him for the purpose of determining the frequency with which second attacks occur in the various diseases below men- tioned. The result is as follows : Small-pox Scarlet fever Measles Typhoid fever Cholera These figures support the view generally entertained by physicians that second attacks of scarlet fever and of measles are comparatively rare, while second attacks of small-pox are not infrequently observed. Considering the very large number of cases of typhoid fever which occur annually in all parts of Europe and America the number of second attacks collected does not bear a very large proportion to the total number taken sick, although the recorded cases, of course, fall far short of the total number of second attacks of this and the other diseases mentioned. The second attacks of cholera recorded are not numer- ous, and, no doubt, a carefully conducted investigation made in the areas of endemic prevalence of this disease would show that second attacks are more common than is indicated by these figures. Second Attacks. Third Attacks. Fonrth Attacks. Total 505 9 514 29 4 33 36 1 37 202 5 1 208 29 3 2 34 36 IMMUNITY AND SERUM-THERAPY. The experimental evidence relating to protective in- oculations in infectious diseases dates from the discovery by Jenner (1768) of the protection afforded against small- pox by vaccination with lymph taken fi'om the vesicles of cow-pox. To Pasteur must be accorded the credit of having first shown by the experimental method that animals may be made immune against other infectious diseases than the one mentioned by inoculations with an " attenuated vi- rus." Commencing- with his experiments upon chicken cholera, in 1880, we shall briefly trace the development of our knowledge up to the present date. Having demonstrated that the disease of fowls known as chicken cholera is due to a sjiecific microorganism, which he was able to cultivate in artificial media, Pas- teur discovered that his cultures became " attenuated " as to their pathogenic power when they had been kept for some time in the laborator}", and that fowls inoculated with these attenuated cultures suffered a comparatively mild and non-fatal attack of the disease, and were subse- quently immune against the pathogenic action of the most virulent cultures, or against contracting the disease by contact with other fowls sufi'ering from it. Pasteur at once comprehended the importance of this discovery, and inferred that what was time of one infec- tious germ - disease was likely to be true of others. Subsequent researches, by this savant, and by other bac- teriologists, have justified this anticipation ; and the experimental demonstration has been made in a consider- able number of similar diseases. Pasteur first obtained an attenuated virus for his pro- tective inoculations against chicken cholera by keeping his cultures for a considerable time freely exposed to the ACQUIKEI) IMMUNITY. 37 air, and ascribed the attenuation to the action of atmos- pheric oxygen. He found that when cultures were made from the blopd of fowls which died from a chronic form of the disease, they possessed an exceptional virulence which was not lost when the cultures were renewed at short intervals ; but that by keeping- these cultures for two months the virulence was greatly diminished, and fowls usually recovered when inoculated with such cult- ures. When kept still longer, his cultures finally lost all pathogenic power. In subsequent exiDeriments with the bacillus of anthrax, Pasteur found that the spores of this bacillus retain their virulence for years, and that it was necessary to exclude them from cultures which were to serve for protective inoculations. When cultivated at a temperature of 42° to 43° C, this bacillus does not form spores ; and Pasteur ascertained that cultures kept at this temperature for eight days no longer killed suscep- tible animals, and could be used in his protective inocu- lations. Other methods of attenuating the virulence of patho- genic bacteria have since been discovered. Thus, Tous- saint has shown that exposure for a short time to a tem- perature a little below that which destroys the vitality of the pathogenic microorganism, modifies the virulence of a culture so that it may serve for protective inoculations. In the case of Bacillus anthracis, Chauveau has shown that a temperature of 50° C, maintained for eighteen minutes, answers the i3urpose. Attenuation of virulence may also be effected by expos- ure to certain antiseptic agents. This was first ascer- tained by the writer in experiments made in 1881, the object of which was to determine the comparative value of various disinfecting agents. Incidentally the fact was 38 IMMUNITY AISTD SERUM-THERAPY. brought out that agents which do not completely destroy the vitality of a pathogenic microorganism may cause an attenuation of its pathogenic virulence. In the experi- ments referred to, the blood of a rabbit recently dead from a form of seiDticoemia induced by the subcutaneous injection of my own saliva, and due to the presence of a micrococcus (Micrococcus pneumoniae crouposse), was subjected to the action of various chemical agents, and subsequently injected into a rabbit to test the destruction of virulence. In the published report of these experi- ments the following statement is made : " The most important source of error, however, and one which should be kept in view in future experiments, is the fact that a protective influence has been shown to result from the injection of virus, the virulence of which has been modified, without being entirely destroyed, by the agent used as a disinfectant. " Sodium hyposulphite and alcohol were the chemical reagents which produced the result noted in these expe- riments ; but it seems probable that a variety of antisej)- tic substances will be found to be equally effective when used in proper proportion." Similar results have since been reported by European bacteriologists. Thus, Chamberland and Roux (1883) ascertained that the anthrax bacillus could be attenuated by adding to cultures certain antiseptic agents — carbolic acid, 1 to 800 ; bichromate of potash, 1 to 100. Another method of attenuating the virus of a patho- genic microorganism is that recently (1892) discovered by Brieger, Kitasato, and Wassermann. This consists in the cultivation of pathogenic bacteria in a bouillon made from the thymus gland of a calf. It was found that the tetanus bacillus cultivated in this bouillon did not form ACQUIRED IMMUNITY. 39 spores, and had comparatively little virulence. Mice or rabbits inoculated with it in small doses — 0.001 to 0.2 c.c. for a mouse — proved to be subsequently immune. And the blood-serum of an immune rabbit injected into the peritoneal cavity of a mouse (0.1 to 0.5 c.c.) was found to g-ive it immunity from the pathogenic action of a virulent culture of the tetanus bacillus. Similar results were ob- tained with several other pathogenic bacteria cultivated in the thymus bouillon — spirillum of cholera, bacillus of diphtheria, typhoid bacillus. Attenuation of virulence may also be effected by culti- vating the anthrax bacillus in the body of a non-suscepti- ble animal, like the frog (Lubarsch, Petruschky) ; or in the blood of the rat (Behring) ; by exposure to sunlight (Arloing) ; and by compressed air (Chauveau). It is a matter of common laboratory experience that many pathogenic bacteria become more or less attenuated when cultivated for a considerable time in artificial media, even when the cultures are renewed at short inter- vals. This is true of the micrococcus of pneumonia, of streptococcus pyogenes, of the bacillus of diphtheria, of the spirillum of cholera, and, to some extent, of the tu- bercle bacillus. Indeed, as a general rule, pathogenic bacteria exhibit greater virulence when cultivated in favorable media, and when recently obtained from the body of a susceptible animal ; and, on the other hand, pathogenic virulence is diminished by cultivation under unfavorable conditions. Probably similar circumstances produce those differences in the type of epidemic dis- eases, as to malignancy or comparative mildness, which have been frequently noted ; external conditions unfavor- able to the development of the specific infectious agent causing an attenuation of virulence, and the reverse. As 40 IMMUNITY AND SERU3I-TIIERAPY. pathog'enic virulence depends, to a considerable extent at least, upon tlie formation of toxic substances during the active develoi^ment of the pathogenic micro-organism, we infer that diminished virulence is due to a diminished production of these toxic substances. An imi^ortant step was made in the progress of our knowledge in this field of research when it was shown that animals may be made immune against certain infec- tious diseases by inoculating them with filtered cultures, containing the toxic substances just refen-ed to, but free from the living bacteria to which they owe their origin. The first satisfactory experimental evidence of this im- portant fact was obtained by Salmon and Smith in 1886. These bacteriologists succeeded in producing an immu- nity in pigeons against the pathogenic effects of the bacillus of hog cholera, which is very fatal to these birds, by inoculating them with sterilized cultures of the bacil- lus mentioned. Similar results were reported by Eoux, in 1888, from the injection into susceptible animals of sterilized cultures of the anthrax bacillus, and also of the bacillus of symptomatic anthrax. More recently (1890) Behring and Kitasato have shown that animals may be made immune against the pathogenic action of the bacil- lus of tetanus or the bacillus of diphtheria bj^ the injec- tion of filtered, germ-free cultures of these bacilli. Sim- ilar results have been obtained by G. and F. Klemperer (1891), in experiments upon rabbits, with filtered cultures of the micrococcus of croupous pneumonia. In Pasteur's protective inoculations against h3'dropho- bia it is probable that the immunity which is developed after infection by the bite of a rabid animal is due to the toxin (toxalbumin ?) of this disease present in the emul- sion of spinal cord which is used in these inoculations. ACQUIRED IMMUNITY. 41 There is also some evidence to show that a certain de- gree of immunity ag-ainst tuberculosis may be produced in g-uinea-pig-s by injections of the toxic substances de- veloped during the growth of the tubercle bacillus — Koch's tuberculin. Evidently the facts stated have an important bearing upon the rationale of acquired immunity, and they ap- pear to support the explanation offered by the writer in a iiaper published in The American Journal of the Medical Sciences in 1881, namely, that immunity depends upon an acquired tolerance to the toxic products of pathogenic bacteria. In the paper referred to I say : " This explanation is, I believe, to be found in the pecul- iar properties of the protoplasm, which is the essential framework of every living organism. The properties re- ferred to are : The tolerance which living protoplasm may acquire to certain agents Avliich, in the first instance, have an injurious or even fatal influence upon its vital activity, and the property which it possesses of transmit- ting its peculiar qualities, inherent or acquired, through numerous generations, to its offshoots or progeny. " There can be but little doubt that protoplasm is the essential living portion of the cellular elements of ani- mal and vegetable tissues, but as our microscoj^ic analy- sis of the tissues has not gone beyond the cells of which they are composed, and is not likely to reveal to us the complicated molecular structure of the protoplasm upon which, possibly, the properties under consideration de- pend, it will be best for the present purpose to limit our- selves to a consideration of the living cells of the body. These cells are the direct descendants of pre-existing" cells, and may all be traced back to the sperm-cell and germ-cell of the parents. Now, the view which I am en- deavoring to elucidate is, that during a non-fatal attack of one of the specific diseases the cellular elements im- plicated which do not succumb to the destructive influ- 42 IMMUNITY AND SERUM-THERAPY. ence of the poison, acquire a tolerance to this poison which is transmissible to their proo-eny, and which is the reason of the exemption which the individual enjoys from future attacks of the same disease. " The known facts in regard to the hereditary transmis- sion, by cells, of acquired properties, make it very easy to believe in the transmission of such a tolerance as we imagine to be acquired during the attack, and if it is shown by analogy that there is nothing improbable in the hypothesis that such a tolerance is acquired, we shall have a rational explanation, not of heredity and the mysterious proi^erties of jjrotoplasm, but of the p^irticu- lar result under consideration. " The transmission of acquired properties is shown in the budding and grafting of choice fruits and flowers, produced by cultivation, upon the wild stock from which they originated. The acquired properties are trans- mitted indefinitely, and the same sap which on one twig nourishes a sour crab-apple, on another one of the same branch is elaborated into a delicious pippin. "Numerous examples in illustration of the same fact may be drawn from the animal kingdom ; thus, the same mother may give birth to two children by different fathers ; the one may inherit a predisposition to con- sumption, and the other to insaniity ; and this inheri- tance, which only manifests itself at the end of many years, has been transmitted from the original sperm-cells of the respective fathers, through countless generations of cells which have lived and died, leaving- their progeny to perform their functions. "The immunity which an individual enjoys from any particular disease must be looked upon as a power of resistance possessed by the cellular elements of those tissues of his body which would yield to the influence of the iDoison in the case of an unprotected person. There is every reason to believe that it is upon the living por- tion of the tissues, or the protoplasm of the body, that the disease-poisons act ; for if it were upon non-living ACQUIRED IMMUNITY. 43 matter — formed material, Beale — and we had to deal only with chemical phenomena, it would be impossible to ac- count for the fact that like causes do not always produce like results. On the other hand, the resistance of living- matter to certain destructive influences is a property dependent upon vitality. Thus, living- protoplasm resists the action of the bacteria of putrefaction, while dead pro- toplasm quickly underg-oes putrefactive changes. Again, it seems probable that in conditions of debility from age, , sickness, starvation, or any other cause, the vital resist- ing- power of the protoplasm is reduced, and certain agents which, under more favorable conditions, would be powerless for harm, may overcome this vital resist- ance. "The tolerance to narcotics, opium, tobacco, etc., re- sulting from a gradual increase of dose, may be cited as an example of acquired tolerance by living- protoplasm to poisons, which at the outset would have been fatal in much smaller doses. There can be little doubt that in this instance it is the living protoplasm of the nervous tissues upon which the poison acts to produce its char- acteristic effects. " But it is in the specific diseases in which a single at- tack proves protective that I find the best proof that the cellular elements of the body may acquire a tolerance during the attack which, being transmitted to their cellu- lar progeny, furnishes the protection which the individ- ual enjoys. "Let us take a particular case. In yellow fever the immediate effect of the poison seems to be to arrest vital processes generally — nutrition, secretion, excretion — and in fatal cases we find that the protoplasm of various or- gans and tissues has undergone degenerative changes ; this is especially true of the liver-cells. Now, we have every reason to believe that this occurs in a less degree in non -fatal cases, but that a sufficient number of cells having resisted the destructive influence of the poison, and become accustomed to its presence, resume their 44 IMMUNITY AND SERUM-THERAPY. functions, and that thus the vital processes upon which the life of the individual depends are again carried on in the very presence of the poison, Avhich at first paralyzed or destroyed the vital activity of certain cells. The case is more striking- in small-pox, in which there is an un- doubted increase of the poison in the tissues during the progress of the disease, but in the first-mentioned disease the patient commonly remains during his sickness in the infected atmosphere, the breathing of which i3roduced the attack from which he is suifering. " The protection from yellow fever resulting from ac- climation — if, indeed, there is such a thing as acclimation independent of an attack of the disease — seems 'to be a tolerance acquired by repeated exposure to the poison in quantities not sufficient to produce an attack. " The tolerance enjoyed by the negro race to the mala- rial poison is probably the result of long residence in malarious regions. Natural selection has doubtless come into play here in establishing this tolerance as a race peculiarity. "I would, then, place acclimation, inoculation by at- tenuated viruses, and an attack of any one of the specific diseases, all in the same category, so far as the explana- tion of the protection afforded is concerned ; and, accord- ing to my view, the explanation of this phenomenon is to be found in the peculiar properties of living proto- plasm which enable it, within certain limits, to adapt itself to varying conditions and injurious influences, and to transmit the impression or modification received in so doing to its offshoots, which continue to perform its functions during the life of the individual." In my chapter on " Bacteria in Infectious Diseases," in " Bacteria," published in the spring of 1884, 1 say : " It may be that the true explanation of the immunity afforded by a mild attack of an infectious germ-disease is to be found in an acquired tolerance to the action of a ACQUIRED IMMUNITY. 45 chemical poison produced by the microorg-anism, and consequent ability to bring the resources of nature to bear to restrict invasion by the parasite." The resources of nature supposed to be brought to bear in restricting invasion by the parasite are referred to on i^age 15 ; the explanation offered being the same as that subsequently known as the Metchnikoff theory. As shown in the discussion of natural immunity, the leucocytes undoubtedly play an important part in the protection of the individual against invasion by patho- genic bacteria. The experiments of Metchnikoff and others show that the introduction of pathogenic bacteria into the subcutaneous tissues of an immune animal often leads to an emigration of leucocytes to the jDoint of inva- sion far exceeding that which occurs in one which has not a natural or acquired immunity for the particular microorganism thus introduced. This fact, however, does not demonstrate the truth of the theory of phagocy- tosis, and, as we shall see, recent researches indicate that the principal factor in the i^roduction of acquired immu- nity is the presence in the blood of the immune animal of some substance capable of neutralizing the toxic i^rod- ucts of the particular pathogenic microorganism against which immunity exists, or of destroying the "germ" itself. These substances are called antitoxins. As pointed out by Buchner in a recent i^aper ((>/?. cit., p. 24), the an- titoxins differ essentially from the so-called alexins, to which natural immunity is ascribed. The alexins are characterized by their germicidal and globulicidal action — they destroy both the red corpuscles and the leuco- cytes of animals belonging to a different species from that from which they have been obtained, and by their 46 IMMUNITY AND SEEUM-THEKAPY. coagulability and instability — destroyed by sunlight and by a temperature of 50° to 55° C. On the other hand, the antitoxins best known (diphtheria and tetanus) have no germicidal or globulicidal action ; they resist the action of sunlight and require a temperature of 70° to 80° C. for their destruction. Our knowledge of the antoxins dates from the experi- ments made in the Hygienic Institute of Tokio, by Ogata and Jasuhara, in 1890. These bacteriologists discovered the important fact that the blood of an animal immune against anthrax contains some substance which neutral- izes the toxic products of the anthrax bacillus. When cultures were made in the blood of dogs, frogs, or of white rats, which animals have a natural immunity against anthrax, they were found not to kill mice inocu- lated with them. Further experiments showed that mice inoculated with virulent anthrax cultures did not suc- cumb to anthrax septicaemia if they received at the same time a subcutaneous injection of the blood of an immune animal. Further, it was found that mice which had sur- vived anthrax infection as a result of this treatment were immune at a later date (after several weeks), when inocu- lated with a virulent culture of the anthrax bacillus. In the same year (1890) Behring and Kitasato discovered that the blood of an animal which has an acquired immu- nity against tetanus or diphtheria, when added to a viru- lent culture of one or the other of these bacilli, neutral- izes the pathogenic power of such cultures, as shown by inoculation into susceptible animals. And also that cult- ures from which the bacilli have been removed by filtra- tion, and which kill susceptible animals in very small amounts, have their toxic potency destroyed by adding to them the blood of an immune animal, which is thus ACQUIRED IMMUNITY. 47 directly proved to contain an antitoxin— wliicli compara- tive experiments show not to be present in the blood of non-immune animals. In the experiments of Behriug- and Kitasato referred to, it was found that 5 c.c. of serum from the blood of an immune rabbit, mixed with 1 c.c' of a virulent filtrate of the tetanus bacillus, and allowed to stand for twenty-four hours, completely neu- tralized its toxic power, as shown by inoculations in mice ; 0.2 c.c. of this mixture injected into a mouse Avas without eiiect, while 0.0001 c.c. of the filtrate, without such admixture, was infallibly fatal to mice. The mice inoculated with this mixture remained immune for forty or fifty days, after which they gradually lost their immu- nity. The blood or serum from an immune rabbit, when preserved in a dark, cool place, retained its power of neutralizing the tetanus toxalbumin for about a week, after which time it gradually lost this power. Behring and Kitasato have also shown that the serum of a diph- theria-immune rabbit destroys the potent toxalbumin in diphtheria cultures. It does not, however, possess any germicidal power against the diphtheria bacillus. Tizzoni and Cattani (1891) have obtained similar re- sults. By repeated inocidations with gradually increas- ing doses of the tetanus toxin they succeeded in pro- ducing- immunity in dogs and other animals ; and they found by experiment that a small amount of blood- serum from an immune dog completely destroys the toxic power of a filtrate from cultures of the tetanus bacillus — one or two drops of serum neutralized 0.5 c.c. of filtrate after fifteen or twenty minutes' contact. They also ascertained that small amounts of blood-serum from an immune dog injected into Avliite mice produced im- munity in these animals. In a subsequent paper, pub- 48 IMMUNITY AND SERUM-THERAPY. lislied in the same year (1891), the authors named re- ported that the tetanus antitoxin in blood-serum from an immune dog- is destroyed by exposure to a temperature of 68° C. for half an hour, or by contact with acids and alkalies ; also that it does not pass through a dialyzing- membrane, Kitasato in the same year (1891) published his impor- tant researches upon immunity from tetanus. He pro- duced an immunity in rabbits, which lasted about two months, by inoculating- them with the filtrate from a culture of the tetanus bacillus, and subsequently, in the same locality, with 3 c.c. of a one per cent, solution of terchloride of jodine ; this last solution was injected subcutaneously in the same dose at intervals of twenty- four hours for five days. Kitasato also ascertained that a small quantity of blood (0.2 c.c.) from an immune rab bit, when injected into the abdominal cavity of a mouse., gave it immunity from the effects of inoculations with the tetanus bacillus. He also made the important dis- covery that mice which were inoculated with a virulent culture of the tetanus bacillus, and which subsequent- ly, after tetanic symptoms had apjieared, received in the cavity of the abdomen an injection of blood-serum from an immune mouse, were preserved from death. The power of the blood of an immune animal to neutral- ize the tetanus poison was also shown by mixing the fil- trate from a virulent culture with blood-serum from an immune animal, and allowing it to stand for twenty -four hours ; a dose three hundred times greater than would have sufficed to kill a mouse proved to be without effect after such admixture with blood-serum. Control experi- ments showed that blood-serum from animals not im- mune had no effect upon the virulence of the filtrate from ACQUIRED IMMUNITY. 49 tetanus cultures. Tlie duration of immunity induced in this way was found to be from forty to fifty days. Yaillard (1891) lias succeeded in producing immunity in rabbits by repeated injections into the circulation of filtered cultures which had been exposed for an hour to a temperature of 60° C. At a temperature of 65° C. both the toxic and the immunizing- action of the filtrate were destroyed. In 1891 G. and F. Klemperer published an important memoir, in which they give an account of their re- searches relating to the question of immunity, etc., in animals subject to the form of septicaemia produced by the micrococcus of croupous pneumonia. They were able to produce immunity in susceptible animals by introduc- ing into their bodies filtered cultures of this micrococcus, and proved by experiment that this immunity had a du- ration of at least six months. They also arrived at the conclusion that the immunity induced by injecting fil- tered cultures into susceptible animals is due to the pro- duction of an antitoxin in the body of the animal. Emmerich, at the meeting of the International Con- gress for Hygiene and Demography, in London (1891), reported results corresponding with those obtained by G. and F. Klemperer as regards the i)roduction of im- munity. He also gave an account of experiments by Donissen in which the injection of 20 to 25 c.c. of blood or expressed tissue juices, filtered through porcelain, from an immune rabbit into an unprotected rabbit, sub- sequently to infection by a bouillon culture of " diplococ- cus lineumonise," prevented the development of fatal septicaemia. Similar results have been reported by Em- merich and Fawitzky in experiments made upon mice with the bacillus of hog erysipelas (rothlauf). 50 IMMUNITY AND SERUM-THERAPY. Brieg-er, Kitasato, and Wassermann have reported (1892) their success in conferring- immunity upon guinea- pigs against the pathogenic action of the cholera spiril- him. They found that attenuated cultures suitable for use as *' vaccines " could be obtained by cultivating- the spirillum in bouillon made from the thymus gland of the calf, by which means they have also obtained attenuated cultures of the bacillus of diphtheria, the bacillus of typhoid fever, the bacillus of tetanus, and the strejjto- coccus of erysipelas. Guinea-pigs inoculated with a cult- ure in thymus bouillon, which had been subjected to a temperature of 65° C. for fifteen minutes, were found, after twenty -four hours, to be immune against virulent cultures in twice the amount which would otherwise have been fatal. During the jiast two or three years numerous addi- tional experiments have been reported which confirm the results already referred to, and show that immunity may be i^roduced in a similar manner against the toxic products of various other pathogenic bacteria — the typhoid bacillus, the " colon bacillus," streptococcus pyo- genes, staphylococcus pyogenes aureus and albus, etc. The Italian investigators Tizzoni and Centanni, in 18&2, published a jireliminary communication in which they gave the results of experiments which appear to show that in guinea-pigs treated with tuberculin, by Koch's method, a substance is developed which neutral- izes the pathogenic potency of the tubercle bacillus. Professor Tizzoni and his associate. Dr. Schwarz, have also (1892) obtained evidence that there is an antitoxin of rabies. Blood-serum taken from a rabbit having an arti- ficial immunity against this disease was found to neutral- ize in vitro the virulence of the spinal marrow of a rabid ACQUIRED IMMUNITY. 61 animal after a contact of live hours. The blood-serum of dogs having an acquired immunity against rabies was found to have a similar action, but in much less deg-ree. The substance (antitoxin) present in the blood-serum of an immune rabbit does not dialyze ; it is precipitated by alcohol, and preserves its activity, to a considerable ex- tent, after precij^itation ; it is soluble in giycerin and is said to have the general chai-acters of a " globulin." The experimenters named also succeeded in conferring im- munity upon susceptible animals by injecting into them blood-serum containing this antitoxin. According to the Italian investigators named, tlie antitoxins of tetanus and of rabies are found only in the blood-serum of immune animals and not in the tissues (nervous or muscular), or in the parenchyma of the various organs. Professor Ehrlich, of Berlin, in 1891, published the re- sults of some researches which have an important bear- ing upon the explanation of acquired immunity, and which show that susceptible animals may be made im- mune against the action of certain toxic proteids of vege- table origin, other than those produced by bacteria ; also that this immunity depends upon the presence of an antitoxin in the blood-serum of the immune animals. The experiments of Ehrlich were made with two very potent toxalbumins, one — ricin — from the castor-oil bean, the otluer — abrin — from the jequirity bean. The toxic potency of ricin is somewhat greater than that of abrin, and it is estimated by Ehrlich that 1 gm. of this sub- stance would suffice to kill one and a half million of guinea-pigs. When injected beneath the skin in dilute solution it produces intense local inflammation, resulting in necrosis. Mice are less susceptible than guinea-pigs, and are more easily made immune. This is most readily 52 IMMUNITY AND SERUM-THERAPY. accomplished by giving them small and graduallj^ in- creasing doses with their food. As a result of this treat- ment the animal resists subcutaneous injections of 200 to 400 times the fatal dose for animals not having this arti- ficial immunity. The fatal dose of abrin is about double that of ricin. When injected into mice in the proportion of 1 c.c. to 20 gm. of body-weight, a solution of 1 i)art in 100,000 of water x)roved to be a fatal dose. The local effects are also less pronounced when solutions of abrin are used. These consist principally of an extensive in- duration of the tissues around the j^oint of injection, and a subsequent falling off of the hair over the indurated area. When introduced into the conjunctival sac, how- ever, abrin prodiices a local inflamrnation in smaller amounts than ricin, a solution of 1 to 800 being sufficient to cause a decided, but temporary, conjunctivitis. Solu- tions of 1 to 50, or 1 to 100, of either of these toxalbumins, introduced into the eye of a mouse, gave rise to a pan- ophthalmitis which commonly resulted in destruction of the eye. But in mice which have been rendered im- mune, by feeding them for several weeks with food con- taining one of these toxalbumins, no reaction follows the introduction into the eye of the strongest possible solu- tion, or of a paste made by adding abrin to a ten per cent, salt solution. Ehrlich gives the following explana- tion of the remarkable degree of immunity established in his experiments by the method mentioned : " All of these phenomena depend, as may easily be shown, upon the fact that the blood contains a body — antiabrin — which completely neutralizes the action of the abrin, probably by destroying this body." In a later paper (1892) Ehrlich has given an account of subsequent experiments which show that the young of ACQUIRED IMMUNITY. 53 « mice which have an acquired immunity for these vege- table toxalbumins may acquire immunity from the inges- tion of their mother's milk ; and also, that immunity from tetanus may be acquired in a brief time by young mice through their mother's milk. In his tetanus experi- ments Ehrlich used blood-serum from an immune horse to give immunity to the mother-mouse, when her young were already seventeen days old. Of this blood-serum 2 c.c. was injected at a time on two successive days. The day after the injection one of the sucklings received a tetanus inoculation, by means of a splinter of wood to which spores were attached. The animal remained in good health, while a much larger control mouse, inocu- lated in the same way, died of tetanus at the end of twenty- six hours. Other sucklings, inoculated at the end of forty-eight hours and of seventy-two hours after the mother had received the injection of blood-serum, like- wise remained in good health, while the control mice died. The possibility of conferring immunity by means of the milk of an immune animal is further shown by the experiments of Brieger and Ehrlich (1892). A female goat was immunized against tetanus by the daily injec- tion of " thymus-tetanus bouillon." The dose was grad- ually increased from 0.2 c.c. to 10 c.c. At the end of thirty-seven days a mouse, which received 0.1 c.c. of the milk of this goat in the cavity of the abdomen, proved to be immune against tetanus. Further experiments gave a similar result, even when the milk of the goat was not injected into the peritoneal cavity of the mouse until sev- eral hours after inoculation with a virulent culture of the tetanus bacillus. When the casein of the milk was separated it retained 54 IMMUNITY AND SERUM-THERAPY. its full immunizing activity, and by concentration in vacuo a thick milk was obtained which had a very high immunization value — 0.2 c.c. of this milk protected a mouse against forty-eight times the lethal dose of a teta- nus culture. In a subsequent communication (1893) Brieger and Ehrlich describe their method of obtaining the antitoxin of tetanus from milk in a more concentrated form. Thej^ found by experiment that it was jDrecipitated by ammo- nium sulphate and magnesium sulphate. From twenty seven to thirty per cent, of ammonium sulphate added to milk caused a precipitation of the greater part of the antitoxin. This precipitate was dissolved in water, dia- lyzed in running water, then filtered and evaporated in shallow dishes at 35° C. in a vacuum. One litre of milk from an immune goat gave about 1 gm. of a transparent, yellowish - white precipitate, which contained fourteen per cent, of ammonium sulphate. This precipitate had from four hundred to six hundred times the potency of the milk from which it was obtained in neutralizing the tetanus toxine. In a still later communication (1893) Brieger and Cohn give an improved method of separating the antitoxin from the precipitate thrown down with ammonium sul- phate. The finely pulverized precipitate is shaken up with pure chloroform, and when this is alloAved to stand the antitoxin rises to the surface while the ammonium salt sinks to the bottom. By filling the vessel to the margin with chloroform, the antitoxin floating on the surface can be skimmed ofi", after which it qnickh' dries. By this method the considerable loss which occurred in the dialyzer, used in the previously described method, is avoided. ACQUIRED IMMUNITY. 55 A most interesting- question presents itself in connec- tion with the discovery of the antitoxins. Does the ani- mal which is immune from the toxic action of any jDartic- ular toxalbumin also have an immunity for other toxic proteids of the same class ? The experimental evidence on record indicates that it does not. In Ehrlich's exper- iments with ricin and abriu he ascertained that an animal which had been made immune against one of these sub- stances was quite as suscejitible to the toxic action of the other as if it did not possess this immunity, i.e., the anti- toxin of ricin does not destroy abrin, and vice versa. As an illustration of the fact, he states that in one experi- ment a rabbit was made immune for ricin to such an ex- tent that the introduction into its eye of this substance in powder produced no inflammatory reaction ; but the subsequent introduction of a solution of abrin, of 1 to 10,000, caused a violent inflammation. In this connection we may remark that there is some evidence to show that persons w^ho are repeatedly stung" by certain poisonous insects — mosquitoes, bees — acquire a greater or less de- > gree of immunity from the distressing local effects of their stings. We have also experimental evidence that animals may acquire a certain degree of immunity from the toxic action of the venom of the rattlesnake. This was first demonstrated by Sewall (1887), and has been recently confirmed by Calmette (1894). In his paper detailing the results of his experiments the author last named says : " Animals may be immunized against the venom of serpents either by means of repeated injections of doses at first feeble and progressively stronger, or by means of successive injections of venom mixed with certain chemi- 66 IMMUNITY AND SERUM-THERAPY. cal substances, among- which I mention especially chlo- ride of gold and the hypochlorites of lime or of soda," " The serum of animals thus treated is at the same time preventive, antitoxic, and therai^eutic, exactly as is that of animals immunized against diphtheria or tetanus." " If we inoculate a certain number of rabbits, under the skin of the thigh, with the same dose, 1 milligr. of cobra venom for example, and if we treat all of these animals, with the exception of some for control, by sub- cutaneous or intraperitoneal injections of the serum of rabbits immunized against 4 milligrs. of the same venom, all of the control animals not treated will die within three or four hours, while all of the animals will recover which receive 5 c.c. of the therapeutic serum within an hour after receiving- the venom." The experimental evidence recorded justifies the con- clusion that, in the diseases referred to, acquired immu- nity depends, chiefly at least, upon the presence of a pe- culiar proteid substance in the blood of the immune animal — antitoxin — which neutralizes the toxic substance — toxin or toxalbumin — to which the morbid phenomena which characterize the disease are due. But it would be premature to infer that in all infec- tious diseases immunity depends upon the production of an antitoxin in the blood of the immune animal. Indeed we have experimental evidence which shows that in cer- tain cases the blood-serum of immune animals has no antitoxic power, but acts upon the germ itself, instead of upon its toxic products. As a rule the antitoxins have no bactericidal action ; but it has been shown by the experiments of Gamaleia, Pfeifi'er, and others, that in animals which have an ac- quired immunity against the spirillum of Asiatic cholera and against spirillum Metchnikovi, there is a decided in- ACQUIRED IMMUNITY. 57 crease in tlie bactericidal power of the blood-serum, and that immunity probably depends upon this fact. The researches of Metchnikoff upon hog cholera, of Issaeif upon pneumonia, and of Sanarelli upon typhoid fever indicate that the immunity conferred upon suscep- tible animals by ijrotective inoculations is not due to an antitoxin but to a substance present in the blood of im- mune individuals which acts directly upon the patho- genic microiJrg-anism, as is the case in cholera-immune animals. The animals immunized are said to be quite as sensitive to the action of the bacterial poisons as are those which have not received protective inoculations. " Their serum does not protect against the toxin, but against the microbe " (Roux). Certain important questions present themselves in connection with the production of antitoxins and germi- cidal substances in the blood of immune animals, one of which is : Is the production of the antitoxin continuous while immunity lasts, or does it occur only during the modified attack which results from inoculation with an attenuated virus, or of filtered cultures, the antitoxin being subsequently retained in the circulating blood ? The latter supposition does not appear very plausible, but it must be remembered that these antitoxins do not dialyze — i.e., they do not pass through animal mem- branes — and consequently would not readily escape from the blood-vessels, notwithstanding the fact that thej^ are held in solution in the circulating fluid. On the other hand, the passage of the tetanus antitoxin into the mother's milk would indicate a continuous supply, other- wise the immunity of the mother would soon be lost. Further experiments are required to settle this question in a definite manner, and also to determine the exact 58 IMMUNITY AND SERUM-THERAPY. source of the antitoxins in the animal body and the modus operandi of their production. According to Buch- ner (1894) the antitoxins are not to be regarded as reac- tive i^roducts developed in the body of the immune ani- mal, but as modified, changed, and " e)it. G97. Resultats des vaccinations charbonneuses pratiques pendant les mois de juillet, aout, et septembre, 1881. Arch, vet., Paris, vii. , 1882, p. 177. De la possibilite de rendrc les moutons refractaires au charbon par la methode des inoculations preventives : avec la collaboration de MM. Cbamberland et Roux. Conipte-rendu, Acad, des Sci., xcii., 1881, pp. 662, 665. Une statistique au sujet de la vaccination preventive centre le charbon, portant sur quatre-vingt-ciuci mille animaux. Ibid., xcv., 1882, p. 1250. Perroucito : Studien iiber Immunitiit gegen Milzbrand. Centralbl. fiir Bakteriol., Bd. V., 1889, p. 50,3. Petermann : Recherches sur I'immunite contre "le charbon. Annales de I'Inst. Pasteur, t. vi., 1892, p. 32. Podmolinoff : Bericht iiber die in 1893 im Chersonlschen Gouvernment ausgefiihrten Milzbi-andschutzimpfungen. Arch. f. Wissencli. u. Prakt. Tierheilk., 1894, p. 805. Serafini e Enriquez : Sull' azione del sangue di animali immuni inocu- lato ad animali suscettibili pel carbonchio. Riforma Med., 1891, No. 152. Toussaint : De I'immunite pour le charbon, acquise A la suite d'inoc- iilations preventives. Compte-rendu, Acad, des Sci., xci., 1880, p. 135. Weyl, Th. : Zur Tlieorie der Immunitiit gegen Milzbrand. Zeitschr. f^ Hygiene, XL, 881, 1892. Wyssokowitsch : Ueber Schutzimpfung gegen Milzbrand in Russlando Fortschr. der Med. 1889, p. 1. II. CHICKEN CHOLERA. Pasteur's researches with reference to the etiolog-y of the disease known in France as cholera de-'^ 2)otdcs, first led him to the discovery that a virnlent culture of a jiatho- genic bacterium may become " attenuated " by certain agencies, and that immunity may be conferred upon sus- ceptible animals by inoculating them with such attenu- ated culture. We now know that his microbe of fowl cholera is a widely distributed bacillus, which is fre- quently encountered in putrefying material, and that it is also extremely fatal to pigeons, pheasants, sparrows, rabbits, and mice. Also that the same, or nearly allied species, may produce an infectious disease of swine (Schweineseuche), of cattle {R'uiderseucJie), and of deer ( Wildseuche). Subcutaneous injection of a minute quantity of a viru- lent culture usually kills chickens within forty-eight hours. Some time before deatli the fowl falls into a somnolent condition, and, with drooping wings and ruf- fled feathers, remains standing in one place until it dies. Infection may also occur from the ingestion of food moistened with a culture of the bacillus, or soiled with the discharges from the bowels of other infected fowls. At the autopsy the mucous membrane of the small intes- tine is found to be inflamed and studded with small hemorrhagic foci, as are also the serous membranes ; the 100 IMMUNITY AND SERUM-THERAPY. spleen is notably enlarged. The bacilli are found in great numbers in the blood, in the various organs, and in the contents of the intestine. In rabbits death commonly occurs in from sixteen to twenty hours, and is often pre- ceded by convulsions. The temperature is elevated at first, but shortly before death it is reduced below the normal. The post-mortem appearances are : Swelling of the spleen and lymphatic glands ; ecchymoses or diffuse hemorrhagic infiltrations of the mucous membranes of the digestive and respiratory passages, and in the mus- cles ; and at the point of inoculation a slight amount of inflammatory oedema. The bacilli are found in consider- able numbers in the blood within the vessels, or in that which has escaped into the tissues by the rupture of small veins. They are not, however, so numerous as in some other forms of septicaemia — e.g.^ anthrax, mouse sep- ticaemia — when an examination is made immediately after death ; later, the number may be greatly increased as a result of post-mortem multiplication within the vessels. The rabbit is so extremely susceptible to infection by this bacillus that inoculation in the cornea by a slight super- ficial wound usually gives rise to general infection and death. This animal may also be infected by the inges- tion of food contaminated wdth a culture of the bacillus. It is by this means that Pasteur proposed to destroy the rabbits in Australia, which have multiplied in that coun- try to such an extent as to constitute a veritable pest. Both in fowls and in rabbits the disease may, under cer- tain circumstances, run a more protracted course — e.g., when they are inoculated with a small quantity of an at- tenuated culture. In less susceptible animals — guinea- pigs, sheep, dogs, horses — a local abscess, without general infection, may result from the subcutaneous injection of CHICKEN CHOLERA. 101 the bacillus ; but these animals are not entirely immune. In the infectious maladies of swine, cattle, deer, and other large animals, to which reference has been made, and which are believed to be due to the same bacillus, the symptoms and pathological appearances do not entirely correspond with those in the rabbit or the fowl ; but the bacillus as obtained from the blood of such animals cor- responds in its morphological and biological characters with Pasteur's microbe of fowl cholera, and Koch's bacil- lus of rabbit septicaemia, and pure cultures from the vari- ous sources mentioned, are equally fatal to rabbits and to fowls. In the larger animals pulmonary and intestinal lesions are developed, and in swine, a diffused red color of the skin, similar to that observed in the disease known in Germany as Schweinerothlauf (Fr. rouget), is some- times seen. According to Baumgarten bacilli from Wildseuche Or from Rlnde7'seuche inoculated into swine give rise to fatal Sclmveineseuche, and bacilli from any of these forms of dis- ease, when inoculated into pigeons, produce character- istic fowl cholera; but 'the bacillus as obtained from Scfiioeineseiiche or Wildseuche is not fatal to chickens, and the bacillus from Schimineseuche is fatal to guinea-pigs, which have but slight susceptibility to the bacillus of rabbit septicaemia. Notwithstanding these differences he agrees with Hueppe in the view that the bacilli from the various sources mentioned are specifically identical ; although evidently, if this view is adopted, we must ad- mit that varieties exist, which differ somewhat in their pathogenic power. In the writer's " Manual of Bacteriology " this bacillus is described under the name Bacillus septicmmice hemor- rhagicce, first proposed for it by Hueppe. In the present 102 I]MM UNITY AND SERUM-THERAPY. cbaiDter we shall give an account of the exiaerimental evi- dence relating- to protective inoculations and serum ther- apy in various animals, with the different varieties of the bacillus in question which have been encountered. It seems probable that the same bacillus was the cause of the fatal form of septicaemia studied by Davaine, and which resulted from the inoculation of susceptible ani- mals with putrefying blood. These experiments by the distinguished French physician constitute an important part of the pioneer work in this field of research. They were commenced in 1868, and are x>ublished in the Bul- letin of the Academy of Medicine {seance of February 18, 1879). Davaine, in the paper referred to, calls attention to the fact, developed by his experiments, that there is a great difference in the resisting iDower of different animals to nie form of septicaemia which had been the subject of his investigations. Thus the rabbit succumbed when inocu- lated with a millionth part of a drop of blood, while guinea-pigs and dogs remained unaffected by such small doses. With reference to the specific cause of the form of septicaemia discovered by him, Davaine says : " The virus is one of the bacteria of putrefaction. I say ' one of the bacteria ' because there is reason to be- lieve that there are among these minute organisms nu- merous species which do not all develop at the same time w^hen they are present in various media." Davaine also discovered the fact that infection de- pends, within certain limits, upon the quantity of bacte- ria introduced into the tissues. He says : " This question of quantity was manifest in our exper- iments. Not only did it vary in different species, the CHICKEN CHOLERA. 103 rabbit and the dog for example, but it may vary iu the same species," The identity of " Davaine's septicaemia " with Pasteur's choUra des ponies is made still more probable by the ex- perimental evidence offered by Toussaint iu a communi- cation to the French Academy of Sciences, made by M. Bouley at the seance of July 25, 1881. In this communi- cation Toussaint says : "Three years ago, July 8, 1878,1 had the honor to present to the Academy an account of a malady due to microbes, which I identified with that studied by Da- vaine in 18G4 and 1865, and which he differentiated from anthrax, for which it had been mistaken by Leplat and Jaillard. " In the month of December, 1878, I made acquaint- ance with fowl cholera, and already, in my thoughts, 1 identified this disease with that which I had observed in \\\y experiments made early in the year. The microbes of the two diseases resembled each other perfectly and behaved the same when inoculated in rabbits. I had, even in 1879, sent to M. Bouley tAvo notes, in which I called attention to the analogies which exist between the parasites of the two diseases and the lesions which they determine, not only in the rabbit but also in pigeons and fowls. " The experiments of the same kind made at the end of 1879 and in 1880 caused me to insert the note published on page 301, vol. xci., of the Convptes-remlvs, under the title of : ' Identity of Acute Experimental Septicaemia and Fowl Cholera.' I gave a resiwie in this note of five series of experiments which had demonstrated to me that inoc- ulations of the microbe of septicaemia give rise to the manifestations of fowl cholera. These results have re- cently been confirmed by additional facts." Toussaint closes his paper by some remarks upon the origin of epidemics of fowl cholera which we quote be- 104 IMMUNITY AND SERUM-TIIEnAPY. cause we believe that the additions made to our knowl- edge of the microbe which causes this disease give sup- port to the views advanced by him in 1881 : " The causes which determine epidemics of fowl cholera are yet unknown. It has been supposed that putrefactive substances may give rise to them, and this has led to the recommendation of cleanliness and disinfection for their prevention. The microbe which kills the first fowl in an epidemic certainly came from some anterior generation which had killed others. But how was it perpetuated ? Do not the facts which demonstrate the development of septicfemia from material undergoing putrefaction throw some light on the question of etiology ? Is it not prob- able that the fowls find the conditions of infection with cholera in the presence of organic matter undergoing pu- trefaction, which may serve as a culture medium for the germs of septicoemia which are in suspension in the air together with the ordinary germs of putrefaction ? " Pasteur's first communication relating to the etiology of fowl cholera was made to the French Academy at the seance of February 9, 1880. In this communication he calls attention to the fact that when fowls are fed with bread or meat soiled with a small quantity of a culture of the microbe of fowl cholera they become infected and their discharges contain the bacillus in large numbers, a fact which readily accounts for the sjjread of the disease in a poultry -yard when a case occurs. In the same communication Pasteur records his obser- vation that " by a certain change in the method of culti- vation the infectious microbe may be caused to have a diminished virulence." Also the fact that fowls inocu- lated with this " attenuated " virus recover and are subse- quently immune against infection by the most virulent mi- crobes. In concluding this communication Pasteur sa3's : CHICKEN CHOLERA. 105 " It appears to be superfluous to point out the princi- pal result of the facts which I have had the honor to pre- sent to the Academy. There are two, however, w^hich it may be useful to mention. These are, first, the hope of obtaining artificial cultures of all kinds of virus ; second, the idea of seeking- for virus vaccines of the virulent mal- adies which have devastated so often, and still devastate, the human race, and w^hich are such a scourge to that branch of agriculture which relates to the breeding of domestic animals." In his communication of October 26, 1880, Pasteur gives his reasons for concluding that attenuation of viru- lence is due to the action upon the microbe of atmos- pheric oxygen. He infers this from the fact, demon- strated by experiment, that when cultures are placed in hermetically sealed tubes, from which the oxygen present is soon exhausted by the growth of the microbe, they do not become attenuated in virulence ; whereas cultures which are freely exposed to the air gradually become attenuated. Pasteur sees in this an impoi-tant fact bear- ing upon the explanation of the natural extinction of epidemics. He says : ^' May we not sujDpose, then, that it is to this influence that we must attribute, in the present as in the past, the limitation of great epidemics." In his communication to the French Academy, made on February 28, 1881, Pasteur treats of the attenuation of virulence by the method above referred to and by the method of Toussaint, and also of the re-establishment of the virulence of attenuated cultures. He says : " The secret of the return to virulence rests solely, at present, upon successive cultures in the bodies of certain animals." 106 IMMUNITY AND SEKUM-TIIERAPY. Thus lie had found by experiment that the anthrax ba- cillus might be so attenuated that it was harmless for grown guinea-pigs, or even for guinea-pigs a month or a week old, but it would still kill guinea-pigs just born — a day old. By inoculating an older pig with the blood of this one, and so on, the virulence was gradually aug- mented, until finally a virus might be obtained which would kill adult animals, and even sheep. In the same way the attenuated microbe of fowl cholera could be re- stored to virulence by first inoculating small birds, such as sparrows or canaries. Applying these facts, demonstrated by his experiments, to the explanation of the origin of epidemics, Pasteur says : " I finished my communication on October 26th by calling attention to the attenuation of viruses by expos- ure to the air as being probably one of the factors in the extinction of great epidemics. The facts presented in this paper, in their turn, may serve to explain the so-called ' spontaneous development ' of these scourges. " An epidemic which has been extinguished by the attenuation of its virus may be reborn by the reinfovce- ment of this virus under certain influences. The accounts which I have read of the spontaneous appearance of the plague appear to me to oft'er examples of this ; for exam- ple, the plague at Benghazi, in 1856-58, the outbreak of which could not be traced. The plague is a virulent malady which belongs to certain countries. In all of these countries its attenuated virus ought to exist, ready to resume its active form when conditions as to climate, famine, and distress again occur. There are other viru- lent maladies which appear 'spontaneously ' in all coun- tries ; such as camp typhoid. Without doubt the germs of the microbes which cause these last-mentioned mal- adies are everywhere distributed. Man carries them CHICKEN CHOLERA. 107 upon him or in liis intestinal canal without great damage, but ready to become dangerous, Avhen, owing to constipa- tion or to successive development upon the surface of wounds, in bodies enfeebled or otherwise, their virulence is progressively reinforced." We believe that the more complete our knowledg"e re- lating to the origin and extinction of epidemics, of the kind referred to by Pasteur, becomes, the more apparent will be the value of his inductions and the clearness of his scientific foresight. Toussaint, on July 25, 1881, reported the results of his experiments upon protecting fowls by a " new method of vaccination." This consisted in inoculating them with the blood of a rabbit which had recently died from septi- caemia produced by the same microbe. As a result of such inoculations the fowls had slight local lesions at the point of inoculation, and soon recovered. They were subsequently found to be immune. Cultures from the blood of a septicBemic rabbit were found to act in the same way. When the culture had been passed throug-h a pigeon, and had then killed a fowl, according- to Tous- saint, it jDreserved its virulence when subsequently passed through the rabbit. Salmon, in the "Keiiort of the Commissioner of Ag^ri- culture " for 1881 and 1882, gives an account of his exper- iments in producing- immunity by the use of a diluted virus. He says : " The experiments of Chauveau, taken with my own, indicate that this method is capable of generalization to the same extent as that discovered by Pasteur ; while the ease and quickness with which the vaccine is prepared, the certainty of effects, the economy of material, and the more perfect protection are points which would appear 108 IMMUNITY AND SERUM-THERAPY. to make it decidedly superior. Wherever the cholera of fowls is raging- a standard cultivation may be made and the vaccine obtained within twenty-four hours ; a single drop of such a cultivation will vaccinate ten, twenty, or even forty thousand fowls, and within three weeks of the commencement of the work the most susceptible of our fowls are insusceptible to inoculations with the strong- est virus. And this, without any sickness, or even local necroses, which Pasteur describes as following vaccina- tions with his attenuated virus." In discussing the practical value of this method Sal- mon estimates the cost as trifling — " not more than half a day's time of one man for one hundred fowls, even if three inoculations were made." In a paper on protective inoculations against fowl chol- era, by Kitt, in the Deidsclte Zeitschrift fi'ir Thiermedichi (December 20, 1886), the conclusion is reached that these inoculations 'undoubtedly protect the fowls from infec- tion either in the natural way or by inoculations with virulent material. But Kitt doubts the practical utility of the method for the arrest of epidemics of this disease in the poultry -yard ; and, as we think, with justice, pre- fers to depend upon cleanliness, disinfection, and prompt removal of infected fowls. As he points out, a consider- able time is required to produce complete immunity, and two inoculations are often insufficient. Pasteur had pre- viously reported that a third inoculation is usually re- quired. But the infection spreads so rapidly when an epidemic is developed in a poultry-yard that a large pro- portion of the fowls would be likely to perish before the protective inoculations could be carried out. Another objection is that when inoculated in the breast muscle the value of the fowl for the table is reduced, and when inoculated in the wing an unpleasant-looking scab is left CHICKEN CHOLERA. 109 at the point of inoculation. The cost in material and time required to carry out the three successive inocula- tions is also an objection to the practical, application of the method. Moreover, the excreta of the inoculated fowls contain the pathogenic microbe, and it would evi- dently be unwise to practise inoculations in poultry- yards not already infected. Kitt states, also, that he has always succeeded in stamping- out the disease very promptly by the other measures referred to — disinfec- tion, cleanliness, separation of all fowls which show any indications of being- infected. In a more recent paper (1893) Kitt reports his success in conferring- immunity upon fowls by a new method, which is, however, rather of scientific interest than of practical value. He first exiierimented to see whether the blood-serum or tissue juices of immune fowls would g-ive immunity against cholera to other fowls, and ob- tained a successful result. He was not, however, able to produce immunity in pigeons or in rabbits by the same method. He next undertook to determine whether the immunizing substance was present in the eggs of fowls which had an immunity as a result of protective inocula- tions. The albumin and yolk of the egg, in doses of 5 to 10 c.c, was injected into the breast of fowls, and at the end of ten days a second inoculation of the same kind was made. Six days after the second inoculation the fowls (five) and a control hen were inoculated with viru- lent blood from a pigeon, and at the same time fed with the chopped-up fiesh and liver of a pigeon just dead from fowl cholera. The control hen died on the following day from typical cholera, the others remained in perfect health. no IMMUNITY AND SERUM-THEKAPY BIBLIOGRAPHY. Gaffky : Die Geflugelcliolera. Zusammenfassender Bericht. Centralbl. fill- Bakteriol., Bd., L, 1887, p. 305. Kitt : Beitrage zur Kenntniss derGefliigelclioleraund deren Scliutzimp- fling. Deutsche Zeitsclir. fiir Tliiermed. iind vergl. Pathol., Bd. XIII., 1886. Eine neue Schutzlmpfunggegen Gefliigelpest. (Gefliigelcholera.) Mtsh. f. prakt. Thierheilk., IV., p. 59. Pasteur, De I'attenuation du virus du cholera des poules. Compte- rendu, Acad, des Sci., xci., p. 673. III. CHOLERA. The spirillum discovered by Koch in 1884 is now pretty generally recognized as the specific cause of Asiatic chol- era. But recent researches indicate that there are nu- merous pathogenic varieties of this spirillum, and show that either an attenuated cholera spirillum or a closely allied saprophyte is not infrequently found in the water of rivers in various parts of Europe. As this spirillum is found in the intestine of cholera patients, and not in the blood, it is evident that its pathogenic action depends upon the chemical products developed during its growth, and this inference is fully justified by the results of experiments upon the loAver animals. These chemical products have been studied by Brieger, Pfeiffer, Sclioll, Gamaleia, Westbrook, and others. Brieger (1887) succeeded in isolating several toxic ptomaines from cultures of the cholera spirillum, some of which had previously been obtained from other sources — cadaverin, putrescin, creatinin, methyl-guyanidin. In addition to these he obtained two toxic substances not previously known. One of these is a diamin, resembling trimethyldiamin ; it gave rise to cramps and muscular tremor in inoculated animals. The other poison reduced the frequency of the heart's action and the temperature of the body in the animals subjected to experiment. In more recent researches made by Brieger and Frankel 112 IMMUNITY AND SERUM-TIIERAPY. (1890), a toxalbumin was obtained from cholera cultures which, when injected subcutaneously into guinea-pigs caused their death in two or three days, but had no effect upon rabbits. Pfeiffer has more recently (1892) published his ex- tended researches relating to the cholera poison. He finds that recent aerobic cultures of the cholera spirillum contain a specific toxic substance which is fatal to guinea- pigs in extremely small doses. This substance stands in close relation to the bacterial cells, and is perhaps an in- tegral part of the same. The spirilla may be killed by chloroform, thymol, or by desiccation, without apparent injury to the toxic potency of this substance. It is de- stroyed, however, by absolute alcohol, by concentrated solutions of neutral salts, and by the boiling tempera- ture, and secondary toxic products are formed which have a similar pathogenic action but are from ten to twenty times less potent. Similar toxic products were obtained by Pfeiffer from cultures of the Finkler-Prior spirillum and from spirillum Metchnikovi. SchoU (1890) took advantage of the fact, previously de- monstrated by Hueppe, that cultures of the cholera spiril- lum in egg-albumin, in the absence of oxygen, are more toxic than ordinary bouillon cultures. Cultures were made by Hueppe's method in hen's eggs. No poisonous pto- maines were found, but two toxic albuminous substances were obtained. The albuminous liquid from the egg cult- ures was dropped into ten times its volume of absolute alcohol, which caused a white precipitate, a portion of which sank to the bottom while another portion floated on the surface. The portion which floated was easily dissolved in a very dilute solution of potash and could be precipi- tated from this solution by the careful addition of acetic CHOLERA. 113 acid, but dissolved in an excess of this acid. It dissolved also in a seven per cent, salt solution, but was precipi- tated by a saturated solution. It g-ave the biuret and xanthoprotein reaction. This substance proved to be very- poisonous. It killed guinea-pigs within twenty minutes when a few cubic centimetres of the alkaline solution — potash — was injected into the cavity of the abdomen. SchoU calls this substance cholera-toxo-globulin. The precipitate which fell to the bottom of the receptacle was washed with alcohol, then digested with water for twenty minutes at 40° C. Very little was apparently dissolved out by this procedure, but this little proved to be very toxic. In from one to three minutes after the injection of a few cubic centimetres of the solution into the peri- toneal cavity of a guinea-pig the animal died. This aqueous solution gave the biuret and xanthoprotein re- action ; it was precipitated by mercuric chloride, nitrate of mercury, and tannin, but not by a saturated solution of ammonium sulphate or acetic acid. This substance Scholl calls cholera-toxo-pepton. The toxic action of these substances is destroyed by a temperature of 100° C, maintained for half an hour, or by 40° to 45° C, main- tained for twenty -four hours. But at ordinary tempera- tures they retain their toxic action for several weeks. Gruber (1892) has also obtained a toxic albuminous precipitate by allowing egg cultures to fall into alcohol, drying the precipitate, and then extracting it with water. Gamaleia (1893) has obtained a toxin which produces the typical phenomena of cholera, which, according to him, is closely associated with the bacterial cells, but can be extracted by a soda solution or by heating to 55° to 60° C. The conclusion is reached that it is a nucleo- albumin analogous to the toxalbumins of tetanus and of 8 114 IMMUNITY AND SERUM-TIIERAPY. diplitlieria. It is precipitated by alcohol, acids, and by magnesium sulphate. Fiuall}^ AVestbrook, in a still more recent research (1894), arrives at the conclusion that the cholera spirillum produces various toxic proteids which in small amounts produce immunity in susceptible animals, and the pro- duction of which depends to a certain extent upon the culture medium ; or that its toxin is a substance of con- stant chemical composition which is mixed with various albuminous substances, either contained in the culture medium or developed in the culture. Duclaux is of the opinion that the last supposition is correct, and that the so-called toxalbumins are not bodies of definite chemical composition, but mixtures of toxins and albuminous sub- stances. Experiments made upon the lower animals show that the introduction of these cholera toxins into the bodj' of a susceptible animal, either with or without the living cholera spirillum, results in the establishing- of a certain degree of immunity against the toxic action of cholera cultures. And there is good reason to believe that a non- fatal attack of cholera in man gives the individual a rela- tive immunity from subsequent attacks, for some time at least. This has led to extended experiments with refer- ence to the possibility of producing a similar immunity in man by means of protective inoculations. The experi- ments bearing upon this point which have been made uiDon the lower animals will first engage our attention. Huejope (1887) first demonstrated the fact that injec- tions of a small amount of a cholera culture into the peri- toneal cavity of a guinea-pig is fatal to these animals. In the following year (1888) Gamaleia reported his suc- cess in infecting guinea-pigs by subcutaneous injections CHOLERA. 115 of blood from an infected pigeon. He found that by suc- cessive inoculations in pigeons a considerable increase in virulence is established, and, that while guinea-pigs were not fatally infected by subcutaneous inoculations with ordinary cultures, they invariably died when inoculated with the more virulent culture in the blood of an infected pigeon. Also, that when guinea-i^igs were inoculated with ordinary cultures, or with cultures sterilized by heat, they were subsequently immune, and resisted inoc- ulations with the most virulent material. In the same year the author referred to announced the discovery of a sj)irillum which closely resembles the cholera spirillum — his " Vibrio Metchnikovi." This was obtained from the intestinal contents of fowls suffering from a fatal in- fectious malady (in Odessa). According to Gamaleia chickens and pigeons which have survived an inoculation with a culture of this spirillum are subsequently immune against the pathogenic action of the cholera spirillum, and vice versa. In subsequent communications Gamaleia re- ported that sterilized cultures of his " Vibrio Metchni- kovi " (sterilized by heat at 120° C) were very pathogenic for rabbits, fowls, pigeons, and even for dogs and sheep. The rabl)it proved to be the most susceptible animal, and succumbed to doses of 4 c.c. in from twelve to twenty hours. Doses of 1 c.c. per 100 gm. of body-weight caused a temporary indisposition followed by immunity. Pigeons were made immune by larger doses. The researches of Pfeiffer (1889) confirmed those of Ga- maleia as to the fact that pigeons and guinea-pigs could be made immune against Vibrio Metchnikovi by the injec- tion of sterilized cultures. But guinea-pigs which had been immunized against this pathogenic spirillum suc- cumbed to cholera infection ; and, on the other hand, ani- 116 IMMUNITY AND SERUM-THEKAPY. mals which had been treated, in various ways with a chol- era culture died without exception when infected with Vibrio Metchnikovi. The conclusion is therefore reached that the two pathogenic spirilla are distinct species, al- thoug"h very similar in many respects. Brieger and Wassermann (1892) have reported the re- sults of experiments with the cholera spirillum cultivated in thymus bouillon. After twenty -four hours develop- ment in this medium the cultures were sterilized by heat (55° C. for fifteen minutes) and placed in an ice-chest for twenty -four hours. Four c.c. of this fluid injected daily for four days into the peritoneal cavity of a guinea-pig made it immune to the cholera spirillum in doses three times as large as were required to kill an animal not so treated. This immunity lasted for two months. Fedoroff (1892) obtained similar results by the subcutaneous injec- tion of sterilized cultures in doses of 1 c.c, in guinea-i^igs. His cultures in thymus bouillon were kept for from seven to ten days at 37° C, then sterilized by heating for fifteen minutes at 65° C, then allowed to stand in a dark room for twenty -four hours, and finally mixed with an equal volume of glycerine. Ketscher (1892) has obtained evidence that the immu- nizing substance in animals which have received protec- tive inoculations is contained in the milk of females thus treated. Three goats received subcutaneous inoculations of virulent cholera cultures, and also injections into a vein and into the peritoneal cavity. The milk of these goats was injected into the peritoneal cavity of rabbits ; these proved to be immune when subsequently lethal doses of a virulent cholera culture were injected into the peritoneal cavity. According to Gamaleia (1892), dogs are ver}^ suscepti- CHOLERA. 117 ble to infection with cholera spirilla, and present symp- toms closely resembling those of cholera in man. They may also be easily immunized against the pathogenic action of cholera cultures. Gruber and Wiener (1892) have also found that suscep- tible animals are easily immunized against cholera infec- tion either by inoculation with small doses, with attenu- ated cultures, or with larger quantities of sterilized cultures. Haffkine (1892) also reports his success in im- munizing guinea-pigs and pigeons. Pawlowsky (1893) claims to have obtained from the blood of animals having an acquired immunity against cholera an antitoxin in the form of an amorphous pow- der ; and Lazarus (1892) reports that the blood of man, after recovery from an attack of cholera, has the property of protecting guinea-pigs from fatal infection when in- jected, in very small amount, into the peritoneal cavity. Issaeff (1894) in an extended series of experiments was not able to entirely confirm the results reported by Laza- rus. In a summary of results obtained in his own experi- ments he says : " 1. The intraperitoneal or subcutaneous injection of blood-serum from normal individuals [that is, persons who have not suffered an attack of cholera], and also of various acids, alkalies, and neutral liquids, gives to guinea-pigs a certain resistance against intraperitoneal cholera infection. This resistance, however, is feeble and temporary, and cannot be considered as identical with the true immunity which results from vaccination with the products of the cholera bacteria. " 2. Guinea-pigs vaccinated against cholera have no immunity against the toxins of the cholera vibrio, not- withstanding their high degree of insusceptibility to in- fection with cultures containing the living vibrio. The 118 IMMUNITY AND SERUM-THEKAPY. blood of immunized guinea-pigs does not possess anti- toxic i:>ropeities. The maximum dose of cholera toxins Avhich immune guinea-pigs can withstand is not greater than that which control animals withstand. "3. The blood of guinea-pigs carefully immunized against cholera possesses si^ecific and very pronounced immunizing, and, in a certain sense, curative powers. "4. The blood of cholera convalescents possesses simi- lar specific and curative x>owers. This property is first developed about the end of the third week after the at- tack, and disappears completely at the end of two or three months." In a series of experiments made by Pfeiffer and Issaeff the results obtained, as stated by Pfeiffer in a subsequent communication, were as follows : " In my research with Issaeff ' uj)on the explanation of cholera immunity ' I proved that the serum of animals which have an active acquired immunity against cholera only has a specific action upon this particular species of vibrio, and as regards other species of bacteria does not difier in its action from the blood-serum of normal ani- mals. We also showed that this specific influence in re- spect to the intraperitoneal cholera-infection of guinea- pigs was due exclusively to bactericidal processes which in some way were induced by the serum of immune ani- mals." The view of Pfeiffer, founded upon his experimental results, is that the destruction of the living cholera spi- rilla, which quickly takes place "in the peritoneal cavity of the guinea-pig, when at the same time a minute quantity of serum from an immune animal is introduced, is not directly due to the bactericidal action of this serum, but that, in some way, it gives rise to a specific bactericidal action in the exudate which is found in the peritoneal CHOLERA. 119 cavity as a result of such injections. His experiments also lead liiin to the conclusion that this is accomplished quite independently of phagocytosis. The brief review of experimental researches relating to cholera immunity which we have made shows that, while there is a general agreement as to the possibility of pro- ducing immunity in susceptible animals, there is consid- erable diiference of opinion as to the true explanation of this immunity. The supposition that it is due to an an- titoxin which has the power of neutralizing the toxic products of the cholera spirillum does not receive any support from the most recent investigations — those of Pfeiflfer and Issaeff — which, on the contrary, seem to es- tablish the fact that this immunity depends upon an in- creased bactericidal activity of the blood-serum of im- mune animals, A very curious fact developed by the researches of the bacteriologists last named is that— " The cholera-serum, which in the peritoneal cavity of guinea-pigs acted only upon the cholera bacteria, and behaved toAvard other vibrios exactly like the serum of normal animals, in a test-tube killed all four species of vibrios with equal rapidity." Unfortunately the evidence relating to the value of pro- tective inoculations in man, although supported by the evidence already referred to as regards the lower ani- mals, is, to a considerable extent, unsatisfactory, owing to the difficulty of applying scientific methods to experi- ments of this kind. The evidence, however, is in favor of the view that a certain degree of protection is afforded by the subcutaneous injection of cholera cultures. Such protective inoculations could not be expected to confer an absolute immunity, inasmuch as the immunity result- 120 IMMUNITY AND SERUM-THERAPY. ing from a single attack has only a relative value, and is probably not of long duration. We quote from Shakespeare's " Rei)ort on Cholera in Europe and India, 1890," the following jaaragraphs re- lating to immunity as a result of an attack of cholera : "immunity after an attack of cholera — EXPERIENCE IN FRANCE, 1884. " The Academy of Medicine of Paris directed a circular letter of questions concerning cholera to the physicians of the localities infected by that disease in 1884, and in group L of general observations in that questonario is found the following : ' Have there been observed recur- rences among the peojDle attacked, either in a former epi- demic or in the present one ? Give the results of this re- currence.' In response to their questions the Academy received 184 communications, but the committee ap- pointed to analyze them eliminated 79 ; for various rea- sons given only 104 were used for analysis. Of this number only 8 bore upon the i:)articular question above mentioned, and it is reasonable to assume that the other 96 observers said nothing concerning this point because they had observed nothing bearing upon it. The results of this analysis may be stated as follows : " From Castelnaudary, with a population of 10,000, we learn that there were 54 cases and 18 deaths from cholera, among which there was 1 recurrence ; from Aix, with 20,- 257, number of cases unknown, deaths 117, among these 2 recurrences were observed, at intervals of ten and forty days ; from Beseges, with 11,400 inhabitants, we learn of 124 cases and 40 deaths, among which were 2 recurrences ; from Cette, with 35,000, the number of cases is not men- tioned, but we learn that there were 92 deaths and 1 re- currence ; from Nantes, with 124,300 inhabitants, we learn of 251 cases and 112 deaths, vvitli 1 recurrence ; from Per- pignan, with 25,000 inhabitants, we hear of 325 cases and 225 deaths, and receive the . indefinite statement that CHOLEEA. 121 there were some fatal recurrences ; from Pignans, popu- lation not stated, we learn of 22 attacks and 12 deaths, with 1 recurrence ; from Cadenet, with a poiDulation of 26,000, we are not informed of the number of cases, but learned that there were 20 deaths and 2 recurrences." " IMMUNITY AFTER AN ATTACK OF CHOLERA — EXPERIENCE IN SPAIN, 1885. "While examining- cholera in Spain, the writer pre- pared a circular containing- a series of twenty-live ques- tions relating especially to the nature, etiology, and proph- ylaxis of cholera, one of which requested the physician to state whether or not, in his own personal experience, he had observed a second or a third attack of cholera dur- ing the same epidemic, and in case of a positive reply to detail the symptoms and all the circumstances surround- ing it. This circular - letter was addressed to some twenty-five hundred Spanish physicians, located in the various cities, towns, and villages in that kingdom which had suffered from the eiDidemic. Among the large num- ber of replies there were only 8 in which a second attack was reported, and from an examination of the details of these there was no doubt left in our mind that 6 were not genuine second attacks after a complete recovery, but were in reality relapses due to imprudences of diet or otherwise before convalescence and complete recovery had been established. Two of the 8 cases, from the details of the reports given, may have been genuine re- current attacks of Asiatic cholera, or may have been sim- ply seizures of cholera morbus (cholera nostras). It is well known that after an attack of Asiatic cholera the digestive apparatus is left in a damaged condition, and disorders of the intestines continue for a long time. The habits of life and the imprudences so common to the class of people most frequently suffering from Asiatic cholera in that country are such as to render them more than usu- ally liable to suffer attacks of cholera nostras. As having an important bearing upon this suggestion, the writer 122 IMMUNITY AND SERUM-TIIEHAPY. made an analysis of tlie vital statistics of Spain, covering the five years previous to 1885, for the purpose of learn- ing the extent of prevalence of cholera nostras among that population, and the result of the inquiry shows that the number of deaths attributed to that disease averaged per year sixteen per every million inhabitants." Dr. Ferran, who practised inoculations on an extensive scale during the epidemic of 1885, in Spain, gives the fol- lowing account of his method of performing these inocu- lations : " 1. The cholera vaccine is nothing more than a pure culture, in bouillon, of the comma bacillus. Its easy and long preservation (four to five days) allows of its trans- portability to great distances, taking care always to keep the flask which contains the material upright. " 2. Heat and cold do not interfere with its preserva- tion if the vaccine is to be used in a short time. It should not, however, be kept out of doors during the warm sea- son. " 3. The vaccine should be kept in flasks of the model of Ferran, with a flat bottom and a short neck. The stop- per, which is of rubber, fits perfectly, and is penetrated by two glass tubes, one straight and short, which does not extend below the inferior surface of the stopper, and which does not project above more than some two centi- metres, is plugged with a small quantity of sterilized cot- ton and a superficial covering of wax. The other glass tube is longer, and extends on the lower side as far as the bottom of the flask, while its superior end is curved, and terminates in a capillary extremity, the tip of which is closed with wax. " 4. When the vaccine is to be used it is necessary to make two principal preparations for the operation. A small syringe for the hypodermic injection, and a small vessel into which it is necessary to empty the fluid from the flask are required. The syringe should have me- CHOLERA. 123 tallic pistons and mountings, witlioiit mastic of any kind and without rubber. Its capacity should be 1 c.c, its needle thicker and shorter than that of ordinary use. Before beginning the vaccination the syringe must be tilled two or three times with boiling water, which is aspirated and expelled through the needle. This is called sterilizing the instrument, and by this means the ex- traneous germs are destroyed which might be contained in it, in order to avoid the production of phlegmons and abscesses. The trouble in taking this precaution will be little. Acting thus, one may perform thousands of injections without fear of any accident. It is sug- gested that it is a bad custom to pass the needle through a Hame in order to sterilize it, because this mode of pro- cedure draws the temper. Another precaution that must be taken relates to the examination of the syringe before using it, in order to be well assured that the piston acts Ijerfectly and that not a single drop of the liquid escapes by a leak in the cannula. This latter defect is sufficient to reject the instrument. If the syringe aspires air because the leather washer, which is placed at the end of the glass tube in order to facilitate its adaptation is dry, or the pis- ton is in the same condition, it is necessary to dela^^ a little while in order to take the syringe apart and soak it in warm water. It is convenient to keep several syringes for use, with a sufficient number of needles, when many inoculations are to be performed. " 5. The small receptacle into which the vaccine is poured in order that the syringe may be filled readily is a capsule, a cup, or some similar vessel. Before use, it should be washed and dried with extreme care, and im- mediately before using passed through an alcohol or Bunsen flame, in order to sterilize it. " 6. All these preparations having been made, the drop of wax which closes the capillary extremity of the long tube of the flask is removed, and at the same time also the wax covering of the cotton-stopper of the short tube, but by no means must this cotton stopper be re- 124 IMMUNITY AND SERUM-THERAPY. moved ; a rubber tube, or the extremity of a small Rich- ardson spray apparatvis, is adjusted to the short tube. The capillary extremity of the loug- tube is now slightly warmed in order to soften somewhat the wax which may have been drawn into its lumen by capillarity, and air is forced into the flask, either by blowing- into the rubber- tube or by working- the Richardson atomizer ; the air in- jected by pressure upon the vaccine fluid forces the latter out through the long tube witli the capillary extremity, and it is collected in the cup or small sterilized vessel. This latter is then covered with white paper, which has been scorched in the flame, or with a sterilized glass plate ; as often as the syringe is filled, this cover will be removed and again immediately afterward replaced. " 7. Never should the rubber stopper which closes the flask, or the cotton which plugs the short straight tube, be removed, because otherwise the germs of the external air might enter and contaminate the culture, and in this way give place to local and general accidents among the inoc- ulated. Whenever, through the movements of transpor- tation, the cotton plug in the short glass tube has be- come so wet as to impede the passage of the air which is to be forced into the flask in the act of expelling the vac- cine from it, it may be removed with the point of a needle and rapidly substituted by another plug of svirgical cot- ton which has been carbonized or salicylized. If this proceeds with cleanness and promptness, there is no dan- ger in doing it. When the cotton, although wet, does not impede the injection of the air, it is better not to change it. " 8. After terminating the vaccination, again the cap- illary extremity of the curved tube is passed through the flame until the small quantity of liquid remaining in it is evaporated ; it is then stopped a second time with a small drop of wax ; and from the other glass tube the rubber tube which has been employed for forcing in the air is removed and another thin layer of wax is placed over the cotton plug. CHOLERA. 125 "9. If in the smaller vessel or cup any of the vaccine fluid remains after the vaccination of all persons present, it is boiled, and in this manner the culture is killed, for it should not be used in another operation, because at- mospheric germs mig-ht become mixed with it. " 10. The technique for the iDractice of the inoculation is the same as for all hyi^odermic injections. The most convenient region is that of the brachial triceps. " 11. The dose is 1 c.c. — or the contents of a syringe — into each arm, for individuals of all ages and conditions. " 12. Five days having elapsed, revaccination may be performed by following the same instructions." Shakespeare, who was sent by the United States Gov- ernment to Spain to investigate the results of these in- oculations, reports as follows : " And now with respect to the human inoculations : The most of these inoculations were performed in villages in the province of Valencia. The number of persons in- oculated considerably exceeds thirty thousand. Much has been both said and written in Spain, France, and Eng- land concerning the results of these inoculations. The results which have been published have appeared to very strongly back up the claim of Dr. Ferrdn that choleraic inoculation has the power of protecting the individual against an attack of cholera, and that the extensive prac- tice of this inoculation among villages already invaded by the epidemic is a powerful and at the same time harm- less means of bringing the epidemic to an end. This being the case, for those who were unwilling to accept the deductions to be made from the published statistics, the only way of escaping their force seemed to be by an attack upon their validity. " The statistics of the anti-choleraic inoculations have been widely attacked. The first public onslaught upon these statistics of which the world, outside of Spain, had much knowledge, was made in the report of the French 126 IMMUNITY AND SEEUM-THEEAPY. Commissiori, with Dr. Brouardel at its head, which "vvas presented to the Minister of Commerce after the return of that Commission from Spain in the summer of 1885. It is charged in that report that the results of the statistics therein reproduced are assailable on account of having- been collected by physicians Avho were partisan support- ers of Dr. Ferran, and that they neither possessed any adequate official character, nor did they possess suffi- cient details. As far as I can learn, the general impres- sion entertained throughout the world of the value of the inoculation statistics is based, in the main, upon this re- IDort of the French Commission. " The statement of that Commission that the statistics which they had been able to obtain of the preventive in- oculations of Ferran were to a considerable degree void of any official character may be true, and perhaps it is also true that they emanated from the jDartisan friends of Ferran; but it must be distinctly remembered that at that day there were practically no official statistics of this kind in the hands of anyone. The official statistics col- lected under the orders of the Spanish Government were gotten together at a far later date. " Ui^on the appointment of the Government at Madrid of the second official Spanish Commission to investigate the Ferran question in the provinces where the inocula- tions were being practised, it was ordered that official statistics of the inoculations should be collected in the usual manner ; that is to say, by the customary statistical officers of the Government. This second medical Com- mission was also accompanied by an independent statisti- cal commission who were charged with the duty of form- ing statistics of those inoculations which were expected to be witnessed by the Medical Commission in their tour of investigation, and the report to the Spanish Govern- ment of this statistical commission is based exclusively upon the official statistics which thej^ themselves col- lected. " In estimating the value of the official character and CHOLERA. 127 the authority of the official statistics, which have since the visit of the F'rencJi Conuidssioa to Spain been collected aud published, the following- circumstances should be taken into account : The provincial governments of Spain are somewhat peculiar, in that the civil governors change with the changes which take place in the Government at Madrid, so that the political constitution of the provin- cial governments is always a reflex of that of the central Government at Madrid. Moreover, the i:)olitical senti- ment of the provincial government is also more or less perfectly reflected by the local governments of the towns of the province. " The hostility of the Minister of the Interior at Ma- drid to Dr. Ferran, and his attempts at the prevention of cholera by inoculation, is a well-known fact now gener- ally admitted ; and the hostility which Dr. Ferran met with from the civil governor of the province of Valencia was even greater than that manifested by the Minister of the Interior himself. " The official statistics of the Ferran inoculations are in the first place signed by the physicians of the locality ; and in the next place by the judge of the municipal court, and sometimes also by the president judge of the judicial district, by the parochial priest, and by the mayor of the municipality, whose signatures and seals are attested by an authorized notary public. " It must, therefore, be obvious that the charge made by the French Commission, which has been so constantly reiterated everywhere, that the public statistics of the anti-choleraic inoculations are void of official character and are to be regarded as ex-parte testimony of the j)arti- sans of Ferran, cannot apply to official statistics which were collected under the supervision of the municijial authorities of the villages wherein the inoculations were performed, and attested not only by the local judicial officers and the parochial priests, but also by the political officers — that is to say, the secretaries and the mayors of the municipalities ; for it must be admitted that neither 128 IMMUNITY AND SERUM-THERAPY. the political officers of the municipalities nor of the pro- vincial governments, any more than the parochial priests, can reasonably be charged with being the partisans or friends of Ferran — the Minister of the Interior continuing during the time of collection of these official statistics to be hostile to the claims of Ferrd,n. It therefore follows that the attack upon the statistics of the inoculations made by the French Commission, and so widely accepted by the medical world as conclusive, does not apply to the official statistics of which we are speaking. And, in view of this fact, the evidence as to the efficiency and harm- lessness of the anti-choleraic inoculations should be re- examined. As I have already said, the results of the preventive inoculations of Ferran, as set forth in the offi- cial statistics, appear to verj^ strongly support his claim of the protective value of the inoculations. In view of the great importance of this whole subject I have deter- mined to place these statistics in this report for the bene- fit of the readers of the English language, in order that they may judge for themselves of the facts as they appear to be recorded. ■" From the Government statistics of cholera throughout the province of Valencia, it appears that among the vil- lages invaded there were 62 attacks per thousand of the population, and 31 deaths per thousand, which gives a mortality of fifty per cent, of those attacked. It appears from analysis of the published official statistics of cholera in twenty-two towns where inoculation Avas performed the inhabitants were divided as follows : 104,561 not in- oculated ; 30,491 inoculated. Of the latter there were 387 attacks of cholera, or 12 per thousand, and 104 deaths, or 8 per thousand ; the mortality of those attacked being twenty-five per cent. Of the former there were 8,406 at- tacks, or 77 per thousand, and 3,512 deaths, or 33 per thousand, being a mortality of those attacked of forty - three per cent. It appears, therefore, that among the population of villages wherein anti-choleraic inoculations had been more or less extensively performed the liabil- CHOLERA. 129 ity of the inoculated to attacks of cholera was 6.06 times less than that of the non-inoculated, whilst the liability of the inoculated to death by cholera was 9.87 times less than that of the non-inoculated. These figures are based exclusively upon the data furnished by inocula- tions, the reinoculations being- left out of consideration, because they are much less numerous, althoug-h from the records of the inoculations it would seem that the liabil- ity of attack, and especially of death by cholera is many times less among them than among those inoculated a single time. " The charge has also been made with resj^ect to the published records of the inoculations that the hygienic and physical condition of the subjects of inoculation have not been sufficiently indicated in the records, and that the vast majority of those profiting by the oppor- tunity to receive the anti-choleraic inoculations were of the middle and upper classes, and therefore not of that class of the inhabitants who are notoriously most liable to attack and death from cholera. This criticism may have some justness as respects some, perhaps many, of the villages where inoculations were performed ; but there are certainly many of the villages wherein the re- sults of the inoculation seemed to be most positively in favor of the claim of Ferran where this criticism can- not hold. I refer to villages wherein three-fourths or four-fifths of the inhabitants were inoculated, leaving only the fraction of the ijopulation non-inoculated. Even in the absence of any special notes indicating the social conditions and hygienic surroundings of the inoculated in these villages it is ridiculous to assume that the vast majority of these were peojile of the middle and upper classes, and were therefore but little liable to attack and death by cholera. Anj^ one acquainted with the char- acter of the Spanish population as it exists in the rural villages, will admit at once that the vast majority of this population consists of the wretched and the poor, who live under the most unhygienic and unsalubrious con- 130 IMMUNITY AND SERUM-THERAPY. ditions, and therefore are of that class most liable to suffer from cholera. " There is still another result of the preventive inocu- lations of Ferrau apparently shown by these statistics. I refer to the apparent marked shortening- of the course of the epidemic after a large percentag-e of the inhabi- tants have become inoculated. It would seem, therefore, from analysis of the official statistics, that the practice of the anti-choleraic inoculation after the method of Ferrau, besides g-iving the subject inoculated a considerable im- munitj' from attack and death by* cholera, furnishes a means of bringing an epidemic rapidly to an end." With reference to Haffkine's method of inoculation we cannot do better than to quote from a lecture which he g-ave in London, in 1893 : " In the research that I have done at the Pasteur Insti- tute on vaccination against Asiatic cholera I have chosen for my starting-point the inoculation of the animal into the peritoneal cavity. Starting from this point I have worked out a method which permits the culture of the microbe in the animal organism in a state of purity during indef- inite generations, the exaltation of it to a well-deter- mined maximum of strength, and keeping it at the same degi-ee of virulence for an unlimited period of time. " This method is illustrated by three series of exjjeri- ments which were the subject of our publications in the Comptes-rendus de la Societe de Biologie of Paris, and which are : " 1. Giving the first animal a dose larger than the fatal dose, and killing this animal in a sufficiently short space of time to be able to find the more resisting microbes. " 2. To expose the exudation taken from the peritoneal cavity to the air" for several hours. " 3. Then to transfer this exudation to the next animal, of large or small size, according to the concentration of the exudation. CHOLERA. 131 "In the hands of a number of other experimenters this method has given the same results and showed a perfect consistency. " The properties of the virus which is obtained in this manner of cultivation are as follows : Upon intraperito- neal inoculation it kills guinea-iDigs regularly in the space of about eight hours, and the fatal dose for this animal is reduced to about twenty times less than that which it would have been necessary to take for the mi- crobe with which I started. The same inoculation kills rabbits and pigeons with a dose which Avould have been perfectly harmless at the beginning of the experiments. It kills guinea-pigs by intramuscular inoculation. "The subcutaneous inoculation brings about the forma- tion of a large oedema, which tends toward sequestration of a whole part of the cutaneous tissues and to the for- mation of a wide open wound, which is cured in from two to three weeks. " The basis of anticholeraic vaccination is founded on the virus obtained in the manner we have just described. " This virus, injected under the skin of a healthy ani- mal, gives it, after several days, immunity from all chol- eraic contamination, in whatever manner this may arise ; that is to say, if an animal that has been thus treated be taken, and an attempt made to infect it either by the di- gestive canal, by neutralization of the gastric juice and the injection of opium into the peritoneum, or by the in- troduction of the microbe into the intestines by the method of Nicati and Rietsch, or by intramuscular inoc- ulation, or finally, by intraperitoneal injection, the most terrible of all, it resists, whilst the control animals suc- cumb. " Anticholeraic vaccination of animals in this manner is then definitely established. But the operation de- scribed cannot be, such as it is, applied to man. The wound following on the subcutaneous inoculation is ter- rible to look at, and, in all probability, extremely painful. Besides, although it does not in itself present any danger 132 IMMUNITY AND SERUM-THERAPY. to the health of the individual, it exposes him to all the complications inseparable from an open wound. " This power of producing necrosis of the cutaneous tissues has been removed from the exalted vaccine by cultivating- it at a temperature of 39° C, and in an atmos- phere constantly aerated. Under these conditions the first generations of the cholera microbe would die rapidly, in an interval of two to three daj's, and therefore care must be taken to sow them again in new media immedi- ately before death, and after a series of generations of this kind a culture is obtained which, if injected under the skin of animals, even in exaggerated doses, only pro- duces a passing oedema, and prepares the organism in such a manner that the injection of exalted virus, the definite vaccine, only produces a local reaction of the slightest description. "vaccination by fixed vacceste. " The method of vaccination thus worked out comprises, then, two vaccines — a mild vaccine, obtained by weaken- ing the fixed virus ; and a strengthened vaccine, which is presented by the virus itself. It is easy to understand why to obtain the weakened vaccine we do not use an ordinary virus, but a virus the nature of which has been previously fixed in the laboratory. It is because the virus, such as is found in the natural state, especially when it has a saprophytic phase of development, presents such pathogenic difterences that there is no certainty in its application. Respecting this we need only recall the story of variolization, and the great danger that an indi- vidual incurred when the infectious substance from a slightly attacked subject was transferred to him. The mildness or the gravity of an infection does not depend only on the veritable strength of the contagious sub- stance, but upon the resistance of the individual fi'om whom it is taken. Thus it happened that in taking vaccine lymph from a subject lightly aflected, a very CHOLERA. 133 weak substance was sometimes produced, whicli was. in- capable of producing' a protective action ; and sometimes a lymph of suck strength that it killed less resistant in- dividuals. The great benefit of Jenner's discovery lay in that it precisely indicated a substance fixed by passages through animals, and of a virulence below that which is fatal to the human organism. Another example is given in the method of Toussaint of vaccination against an- thrax, the first of its kind, which has been obliged to make way for the method of M. Pasteur, for the sole reason that the latter, based upon virus of a fixed nature, presented an absolute ceiiiainty in its results which was wanting in the other. Finally, in the history of cholera itself I may recall the attempt made in 1885 by Dr. Fer- ran, of Barcelona, who, with the object of j) reserving the population of the Peninsula from the epidemic of cholera, made injections in his patients of the ordinary virus taken from dead bodies and cultivated in the laboratory. The statistics of the results obtained by this means showed such uuceiiaintj^ that no one dared to recommend this operation to his country in spite of the very numer- ous tiials made in Spain. " The possibility of treating the animal organism by vaccines of an absolutely fixed nature, prepared by means of special operations, constitutes, on the contrary, the basis of the Pasteurian method, and here lies the whole secret and the sole g-uarantee of the success of its appli- cation. " APPLICATION or THE IVIETHOD TO MAN. " The method of anticholeraic vaccination, worked out by experiments on guinea-pigs, was tried upon rabbits and pigeons before it was applied to man. These ani- mals were chosen in order to have subjects very difi^er- ently organized, and in order to be able to generalize the conclusions, and to be able to extend them to the human organism. *' The result obtained on all these animals being abso- 134 IMMUNITY AND SERUM-THERAPY. lutely the same, it was decided to apply the operation to man. " The symptoms produced by this operation have been described iu several scientific magazines. The method has been tried at Paris, at Cherbourg-, and at Moscow, on about fifty persons of both sexes, between the ages of nineteen and sixty-eight, of French, Swiss, Bussian, Eng- lish, and American nationality. " In every case the method has shown itself absolutely harmless to health, and the symptoms that it evoked were a rise of temj^erature, a local sensitiveness at the place of inoculation, and the formation of a transitory oedema at the same iDlace. The first sensations are felt about two or three hours after inoculation ; fever and general indisposition disappear after twenty - four to thirty-six hours ; the sensitiveness and oedema last, grad- ually dying away in from three to four days. The symp- toms following the second inoculation were generally rather more marked, but of shorter diiration. The whole recalls the sensation of a bad cold in the head, lasting about one or two days. " The microbes introduced under the skin do not jirop- agate, but after a certain time they die and disaj)pear. It is the substances which they contain, and which are set free when they die, that act upon the animal organ- ism and confer immunity upon it. It is found that the same result can be obtained if the microbe be killed be- fore inoculation, and if their dead bodies only be injected. Thus I have been able to prepare vaccines preserved in weak solutions of carbolic acid. In this the microbes die at the end of several hours, and the vaccine so pre- pared has been found still efficacious sis months after its preparation. It is evident that there is much advantage in this state of preservation of the microbes. They can be used by persons having no bacteriological training, and the absence of every living organism makes them perfectly safe. The carbolic acid that they contain pre- serves them against any invasion of other microbes. CHOLERA. 135 Finally, as tliey can be kept for several months, their preparation can be intrusted to a central laboratory, whence the vaccine mn%n)%iles can be sent out to oper- ators. But it may be presumed that immunity given by these preserved vaccines will not equal in jDersistency that produced by living- ones, and as the method is not yet backed up by established statistics, it is better that vaccinations should be done as much as possible with living virus, so as to obtain the most conclusive results. " As to the length of time that immunity produced by living vaccine lasts, we have not yet at the laboratory animals that have been inoculated at a very distant date ; those upon which we experimented dated from, at most, four months and a half. At the end of this time their immunity was found to be still perfect, and we do not despair of its lasting much longer yet. HARMLESSNESS OF THE METHOD. "The inoculations upon man, added to the hundreds of experiments that we have made upon animals, testify to the perfect harmlessness of these operations, and there is no difficulty in proving their efficacy by experiment, no matter on what species of animal. We have taken twelve guinea-pigs, and vaccinated six of them with vac- cines preserved in carbolic acid since September 8th last. Yesterday, at five o'clock, six days after the first vaccina- tion, we injected into the peritoneal cavity of all the non- vaccinated animals a fatal dose of virus, and into the vaccinated animals we injected a double dos^ The six vaccinated animals are perfectly well, while of the others two have already died of choleraic poisoning, two are very ill, and the others will certainly soon become so. But it is evident that T cannot perform a like experi- ment on man (but, however, this would be the only means C)i being able to give a definite experimental demonstra- tion)." 136 IMMUNITY AND SERUM-TIIERAPY. Further details as to the method are given by Wood- head in the " Edmburg-h Hospital Reports," as follows : " In order to be absolutely certain that the virus is pure, M. Hafi'kiue makes cultivations before each inocu- lation of the human subject, by Eoux and Yersin's method, one devised for the separation of the diphtheria bacillus. A small drop of the virus exalte is taken on a spatula- shaped needle, and streak after streak is made with the Hat of this needle on the surface of the agar in the tubes, a couple of tubes being used, so that twelve streaks per- haps, in all, are made without the needle being recharged ; in the earlier streaks, of course, the seed bacilli are so close together that a continuous line of colonies makes its appearance ; but along the course of the later streaks, colonies, with distinct intervals between them, are devel- oped ; part of one of these is examined under the micro- scope, in order to determine that it is made up only of comma bacilli, and then the other part is used for seed material for a tube-culture preparatory to inoculation. " The inoculation itself is an exceedingly simple proc- ess ; the needle and the syringe are boiled ; the tube containing the material to be used for inoculation re- ceives a syringeful or pipetteful of sterilized beef-broth, then with a platinum needle the culture is thoroughly mixed with this broth, so that a kind of emulsion is pre- pared ; this emulsion is drawn up in a sterilized pipette, and is then passed into a sterilized conical glass covered with sterilized paper. If a sixth of the culture is to be introduced, two more syringefuls or pipettefuls of broth are to be added, so that we now have three in all ; if an eighth, three are added, and so on ; the whole is mixed, and then half a syringeful is taken for use for each pa- tient. In inoculating, the skin, just above the crest of the ilium, is thoroughly cleansed with five per cent, solution of carbolic acid, the attenuated virus is inoculated on the left side, and then after an interval of four or five daj's the second vaccine, or the more virulent form, is inoculated CHOLERA. 137 on the right side. After inoculation everything that has been used is thoroug-hly boiled, the skin of the patient is again washed with five per cent, carbolic acid, and the table is washed down with the same solution." Haffkine commenced his experiments on man by inocu- lating himself, and has repeated the inoculation three times. He next inoculated about fifty individuals in Paris, Cherbourg, and Moscow, and demonstrated in a satisfactory way that the inoculations are without danger. A first inoculation in an unprotected person is said to give rise to some malaise and febrile reaction, to pain and tumefaction at the point of inoculation, and swelling of the neighboring glands. The second inoculation with a strong virus, made after an interval of six days, causes also some elevation of temperature, but no swelling at the point of inoculation. This slight reaction from a strong virus is supposed to be satisfactory evidence of a certain degree of immunity as a result of the first inocu- lation. The results of the protective inoculations by Haffkine's method, which have been ijractised in India, indicate that these inoculations have a real value, but that immu- nity is not immediately established, and consequently that during an epidemic a certain number of fatal cases may be expected among the inoculated as Avell as among the non-inoculated. This is illustrated by the results of inoculations made among the prisoners in Gaya jail (1894), reported by Surgeon-Major Macrae, I.M.S., from whose report we quote as follows : " Cholera broke out in the Gaya jail on the 9th of July, and from that date to the 2d August 34 cases oc- curred, with 20 deaths, there being on date of first attack 422 prisoners in jail. The disease was clearly traceable t 138 IMMUNITY ATSTD SERUM-THERAPY. to importation, but its diffusion among the prisoners was a question of much greater difficulty. The sanitary con- dition of the jail is excellent ; it was built quite recently, on the latest plans, and is generally considered a model jail. The water-supply, which is from a well, is of excel- lent quality and protected from pollution, and it is be- lieved that the spread of the disease was largely due to the agency of flies finding access to food and milk after being in contact with cholera poison, and contaminating them. From the 9th to the 17th July, 6 cases occurred, with 5 deaths. " Many of the prisoners on being told about ])reventive inoculation wished to be inoculated, and M. Haffkine, who had previously been communicated with, and whose zeal and enthusiasm in the cause that he so well advo- cates are beyond praise, arrived here on the 18th July, and in the presence of Surgeon-Colonel Harvey, who kindly assisted, and myself, inoculated 147 prisoners, and on the 19th 68, making a total of 215 out of 433 present in the jail on that date. " Being purely voluntary, no selection of prisoners was possible ; but all classes in the jail were represented, male and female, old and young, habituals and less frequent oftenders, strong and weakly, convalescent and even hos- pital patients sent their representatives. No difference of any kind was made between inoculated and non-inocu- lated prisoners ; they were under absolutely identical conditions as regards food, water, accommodation, etc., in short, in every possible respect. " As, owing to the progress of the epidemic, a large number of prisoners were removed from the jail into camp, it will be found convenient to consider the effect produced by the anticholera inoculation under three headings : " (a) The first will include the period from the IStli July, the date of first inoculations, to the 24th July, the date on which final reinoculations were made, and refers to all the prisoners. CHOLERA. 139 " (h) The second concerns the prisoners who remained in jail after the majority were removed into camp, and comprises the period from the 25th July, to the 2d August, on which date the final case occurred among' this body of prisoners. "(c) The tldrd refers to the body of prisoners who were moved into camp on the 25th July, and includes the period between that date and the 1st Aug"ust, when the final case occurred among this body. Averase present. Chol- era. Percent- age of average strength. Deaths. Percent- age of average strength. Percent- age of deaths to cases. Inoculated 211.2 209.0 32.5 48.55 171.42 146.5 5 7 1 7 2 6 No. I. 2.37 3.34 No. II. 3.07 14.42 No. III. 1.16 4.09 4 5 Nil. 3 1 2 1.89 2.39 Nil. 6.18 0.58 1.36 80 Not inoculated Inoculated 71.42 Nil. Not inoculated Inoculated 42.86 50.0 Not inoculated 33.33 " The conclusions to be drawn from the results above recorded appear to me to be that for the first few days the inoculations have scarcely any protective influence ; then their effect seems to gradually increase. M. Haff- kine in his publications has laid stress on the fact that he anticipates a period of ten days would elapse from date of first inoculations before the full effect would be obtained. During the first five days after fi r s t inocula- TION. First three dats after second inocu- LATION. Last six days. Cases. Deaths. Cases. Deaths. Cases. Deaths. Inoculated 5 7 4 5 3 5 1 3 Nil. 8 Nil. Not inoculated 2 140 IMMUNITY AND SERTTM-TIIEKAPY. " Further observations are necessary to prove whether the inoculations as now practised will prove of lasting benelit ; the results obtained in Gaya jail seem to me to justify the conclusion that their temporary beneficial ef- fect is undoubted. "I have been informed by M. Haffkine that he pro- poses to introduce a certain modification of his method, with the object of afibrding- protection to patients during the ten days necessary for the action of his vaccines. I think there is every reason to believe that better results would have been obtained here had the inoculations been performed at an earlier period instead of during the epidemic." In a recent pajaer {British Medical Journal, January 26, 1895) Haft'kine gives the following summary of his inoculations in India : " Table showing the total 7iumher of persons on whom ob- servations have been made in Calcutta, Gaya, Cawnj^ore, ccnd L'uchtiow. Number. Cases. Percentage of Cases to Strength. Percentage Deaths. of Deaths to Strength. Non-inoculated. Inoculated 1,735 500 174 21 10.63 4.20 113 ! 6.51 19 3.80 Total 2,235 195 132 Other methods of producing immunity in man have been proposed, and recent experiments indicate that this may be accomplished through the digestive tract by the ingestion of considerable quantities of sterilized cultures. Thus Klemperer (1892) has obtained results which seem to show that immunity in man may be induced not only by the subcutaneous injection of virulent cultures but also by the subcutaneous injection of the milk of immu- CHOLERA. 141 nized goats and by the ingestion of cultures sterilized by heat. The degree of immunity, as determined by the activity of the blood-serum of the immune individual for the protection of guinea-pigs, is considerably less, how- ever, than when repeated injections of virulent cultures have been made. The blood-serum of individuals made immune by the last-mentioned method is said by Klemp- erer to protect guinea-pigs when injected into the cavity of the abdomen in the dose of 0.005 c.c. And the injec- tion of 5 c.c. of milk from an immunized goat is said to confer such an immunity that 0.25 c.c. of blood-serum from the immune individual is sufficient to protect a guinea-pig from cholera cultures. In this connection attention is called to the results obtained in more recent experiments by Pfeiffer (p. 118). Sawtschenko and Sabolotny (1893), as a result of a se- ries of experiments made upon themselves and laboratory assistants, arrive at the following conclusions : " 1. After the ingestion of sterilized (by heat) and sub- sequently carbolized agar cultures of cholera bacteria the serum of man acquires an immunizing property as regards the cholera vibrio. " 2. As a result of the ingestion of sterilized agar cult- ures the individual is protected from infection with viru- lent cultures of the cholera vibrio by way of the intestine. " 3. The discharges of individuals immune against chol- era, and to all outward appearance in perfect health, may contain a great number of cholera vibrios (in case they are in any way introduced into the intestine) and may thus serve to propagate the malady." SeruT^i-thcrapy . The results of experiments already referred to are fa- vorable to the view that the serum of immune individuals 142 IMMUNITY AND SERUM-THEllAPT. (man or lower animals) contains a substance (antitoxin ?) wliicli would i^robably be useful in the treatment of chol- era if a practical method of obtaining- it in sufficient quantit}^ should be devised. This view receives support from the exj)eriments of Pawlowsky and Buchstab (1893) and of Fedoroff (1893). The first-named bacteriologists, after demonstrating the fact that guinea-pigs, rabbits, and dogs could readily be immunized by the methods hereto- fore refen*ed to, proceeded to make further experiments with the serum of immunized dogs. The serum from these animals proved to have a decided bactericidal ac- tion for cholera cultures, and when injected into the peri- toneal cavity of rabbits or guinea-pigs, in doses of 5 c.c, it made these animals immune against the usually fatal doses of virulent cultures of the cholera spirillum. The dogs were immunized by first injecting attenuated cult- ures subcutaneously in doses of 10 c.c, and in following' this with more virulent cultures and larger doses, until at last the animal received 90 c.c. of a virulent culture. The immunizing value of the serum obtained from a dog treated in this way was estimated at 1 to 130,000. Five c.c. of a virulent culture mixed with 1 c.c. of this serum pro- duced no efi'ect when injected into the peritoneal cavity of guinea-pigs. And the curative value of the serum, w'hen injected subcutaneously two to five hours after in- traperitoneal infection with 5 c.c. of a virulent culture, was shown b}^ the fact that out of 16 animals treated in this way 12 survived, while the control animals all died. The serum was injected by the authors referred to be- neath their own skin in doses of 1 c.c, and proved to have no noticeable effect upon the pulse or temperature. Fedorofl* (1893) did not succeed in obtaining a serum of as high an immunizing- value as that obtained by the au- CHOLERA. 143 thors last referred to, or by Klemperer, but he arrives at the conclusion that " it is a settled fact that animals may- be rendered immune against cholera in a surprisingly short time by means of the blood-serum of other immune animals. Also that blood-serum therapy in Asiatic chol- era seems much more promising than the method pro- posed by Brieger, Kitasato, and Wassermann — injections of cultures in thymus bouillon." Dr. Freymuth, of Danzig, in the Deutsche Medicinische Woche/ischrift of October 25, 1894, reports three cases of cholera treated by subcutaneous injections of blood-serum from individuals who had recently suffered attacks. The first case, a female, was in a complete state of col- lapse on August 13tli, when 10 c.c. of serum was injected under the skin between the shoulders. No improvement was noted as a result of the injection. The following day a dose of 30 c.c. was given in the same way. No im- provement. The following day a dose of 50 c.c. was fol- lowed by apparent improvement, but this was not main- tained, and the patient died on the 18th. In the other two cases recovery occurred. One received in all 80 c.c. of serum, and the other 20. As the reporter observes, the number of cases is too small to justify any conclusions as to the value of the treatment ; but they are of interest as being the first cases reported in which this mode of treatment was resorted to, and as showing, at least, that it is harmless and not difficult to carry out when there are persons at hand who have re- covered from a recent attack, and are Avilling to spare the blood. With reference to the practical value of protective in- oculations for the prevention of cholera epidemics, the writer sees no reason to modify the views expressed in 144 IMMUNITY AND SERUM-TIIEKAPY. his paper on " Protective Inoculations in Infectious Dis- eases," read at the twentieth annual meeting of the American Public Health Association (1892). Speaking of Ferrdn's inoculations, and the more recent exioeriments of Haffkine, I say : "Whether this method will be found to have any great practical value, can only be determined by more extended exiDeriments. But in view of the fact that other measures of prophylaxis, well known to sanitarians, are sufficient for the prevention of cholera epidemics, and that nurses and others who necessarily come in contact with cholera patients are not likely to contract the disease if they use proper precautions with reference to their food and drink, the disinfection of their hands, etc., we doubt whethei protective inoculations will ever come into general use as a measure of prophylaxis against this disease. Cer- tainly they cannot take the place of those sanitary meas- ures which have been proved to be sufficient for the pre- vention of epidemics, namely, exclusion by a proper inspection service at ports of entry (' quarantine '), isola- tion of the sick, disinfection of excreta, general sanitary police of exposed towns and cities, boiling the water used for drinking purposes, etc. Still, under certain circum- stances, i^rotective inoculations may have considerable practical importance, and the experiments now being made have evidently great scientific interest in connection with the question of acquired immunity." BIBLIOGRAPHY. Aviragnet : Die Impfungen gegen die Cholera und die Metbode Dr. Fenan's. Bull. Med., 1892, No. 83. Brieger : Zur Kenntuiss des StofEwechselprodukte des Cholera bacillus. Berl. klin. Wchschr., 1887, p. 817. Brieger, L., und Wassermann : Ueber kunstliche Schutzimpfung von Thieren gegen Cholera Asiatica. Dtsche. Med. Wchschr., 1892, p. 701. CHOLERA. 146 Bruce : Bemerkung uber die Virulenzsteigerung des Cholera vibrio. Centralbl. fiir Bukteriol., Bd. IX., 1891, p. 786. Calmette, A. : Rechercbes experiraentales sur le cholera asiatique indo- chinois, et sur rimmunisation chimique des animaux contre cette maladie. Arch, de Med. navale, 1893, pp. 216, 257. Fedoroflf, S. : Zur Frage von der kunstlichen Immunitat bei Cholera asiatica. (Russian.) Abst. in Baumgarten's Jahresbericht, 1892, p. 345. Zur Blut-serumtherapie der Cholera asiatica. Zeitschr. fiir Hyg., Bd. XV., 423, 1893. Ferran : Revendication de la priorite de la decouverte des vaccius du cholera asiatique, faite sous les auspices de la Municipalite de Bar- celone. Barcelone, 1888. Sur I'actiou pathogene et prophylactique du bacillus-virgule. Compte-reudu, Acad, des Sci,, t, c, p. 959. L'inoculatiou preventive contre le cholera morbus asiatique. 8°, Paris (Soc. d'Ed. scienti.), 1893. Fokker, A. P. : Ueber eiu durch Cholerabacillen gebildetes Enzym. Dtsche. Med. Wchschr., 1892, p. 1151. Freymuth : Drei Cholerafalle, behandelt mit menschlichem Heilserum. Dtsche. Med. Wchschr., 1894, No. 43, p. 829. Gameleia, N. : Recherches experiraentales sur les poisons du cholera. Arch, de Med. experimen., iv.. No. 2. — Ueber Praventiv-impfuiig gegen Cholera asiatica. Compte-rendu, Acad, des Sci. 1888 (Aug. 20th). Sur la vaccination cholerique. Compte-rendu, Soc. de Biol., 1889, No. 38. Vibrio Metchnikovi, vaccination chimique. Ann. de I'lnst. Pasteur, vol. iii., p. 542. — Ueber die Resistenz der Kaninchen gegeniiber den Cholerabaii- terien. Centralbl. fiir Bakteriol., Bd. IX., 1891, p. 807. Gruber, M. : Weitcre Mittlieilungen Uber vermeintliche und wirkliche Choleragifte. Wien. klin. Wchschr., 1892, p. 685. Haffkine : Inoculations de vaccins anticholeriqucs h I'homme. Le Bull. Med., 1892, p. 1113. Vaccination against Asiatic Cholera. Indian Medical Gazette, 1893, p. 97. A Lecture on Anticholeraic Inoculation. British Medical Jour- nal, 1893, p. 278. Hammerl : Tierinfektionversuche mit Cholerakulturen verschiedeuer Herkunft, etc. Hygien. Rundschau, 1893, No. 13. Ilkewitsch, K. J. : Ueber Choleraschutzimpfungen, etc. Wratsch 1893, pp. 785, 814, 858, 889. (Russian.) Issaeflf : Untersuchungen uber die kiinstliche Immunitat gegen Cholera. Zeitschr. fur Hyg., XVI., 287, 1894. 10 146 IMMUNITY AND SERUM-THERAPY. Issaeff und Ivauoff : Untersuchungen uber die Immunisirung der M( er- schweinchen gegeu den Vibrio Ivanoff. Zcitsclir. fiir Hyg., XVII., 117, 1894. Jaweia, G. : Observations sur des cobayes immunises par les vaccins anticholeriques vivants. Annal. de I'lust. Pasteur, 1892, p. 708. Kanthack and Westbrook : Report on Immunity against Cholera. Brit- ish Medical Journal, 1893, p. 572. Ketscher, N. : De I'immunite contre le cholera conferee par le lait. Compte-rendu, Soc. de Biol., 1892, No. 32. Klein, E. : The Anticholera Vaccination ; an Experimental Critique. British Medical Journal, 1893, p. 632. The Anticholera Vaccination ; an Experimental Critique. Indian Medical Gazette, 1893, p. 135. ; Ibid., p. 138. (Klein's critique criti- cised by Haffkine.) Die Anticholera-Vaccination. Centralbl. f. Bakteriol., XIII., 426, 1893. Klemperer, G. : Untersuchungen iiber Schutzimpfung des Menschen gegen asiatische Cholera. Berl. klin. Wchschr., 1892, p. 969. Weitere Untersuchungen iiber Schutzimpfung des Menschen gegen asiatische Cholera. Berl. klin. Wchschr., 1892, p. 1265. Zur Kenntniss der natiirlichen Imraunitat gegen asiatische Choi-' era. Deutsche Med. AVochensclir., 1894, No. 20. Untersuchungen iiber kiinstlichen Impfschutz gegen Cholera- intoxication. Berl. klin. Wchschr., 1892, p. 789. Untersuchunaren iiber Infektiou und Immunitat bei der asia- tischen Cholera, 8°, p. 183. Berlin (August Hirschwald), 1894. Lazarus, A. : Ueber antitoxisclie Wirksamkeit des Blutserums Cliolera- geheilter. Berl. klin. Wchschr., 1892, p. 1041. Macrae, R. : Preventive Inoculation for Cholera in India. British Med- ical Journal, 1894, p. 653. Cholera and Preventive Inoculation in Gaya Jail. Indian Medi- cal Gazette, 1894, No. 9, p. 334. Palmirski : Vibrio Metschnikovi und die Immunisation gegen denselben mittelst Impfung von Choleravibrion und vice versa. Gaz. lekar- ska, 1893, No. 31. (Polish.) Centralbl., XIV., 789. Pawlowsky und Buchstab : Zur Immunitatsfrage und Blutserumthera- pie gegeu Cholerainfektion. Dtsch. Med. Wchschr., 1893, Nq. 22. Pfeiflfer : Untersuchungen iiber das Choleragift Zeilschr. fiir Hygi- ene. Bd. II., 1892, p. 393. Weitere Untersuchimgen uber das Wesen der Choleraimmuni- tat, etc. Zeitschr. fur Hygiene, Bd. XVIII., 1894, p. 1. Pfeiffer und Wassermann : Untersuchungen uber das Wesen der Chol- eraimmunitat. Zeitschr. fur Hygiene, Bd. XIV., 1893, p. 46. Pfeiffer, R. und Issaeff : Ueber die spezifische Bedeutung der Cholera- immunitat. Zeitschr. f. Hygiene, XVII., 355, 1894. CHOLERA. 147 Remesoff, G. N. : Ueber prophylaktische Choleraimpfiingen nacb der V. A. Chawkinscben Metbode. Med. Obozren, 1893, p. 487. (Rus- sian.) Sabolotny, D. : Infektions- und Immunisierungsversucbe am Ziesel (Spermopbilus guttatus) gegen deu Cboleravibrio. Centralbl. f. Bakteriol, XV., 150, 1894. Salus, H. : Ueber das Verbalten der Cboleravibrionen im Taubenkor- per und ibre Beziebuug zum Vibrio Metscbnikovi. Arcb. f. Hy- giene, XIX., 333, 1894. Sawtscbeuko, J., und Sakolotny : Versucb einer Immunisation des Menscben gegen Cbolera. Centralbl. f. Allgem. Patbol., IV., No. 16, 1893. Abst. in Centralbl., XV., 28. Scboll : Ueber das speciflscbe Cboleragift. Internat. klin. Rundscbau, 1890, No. 42, p. 1726. Soberbeim, G. : Experimentelle Untersucbungen iibcr Cboleragift und Cbolerascbutz. Zeitscbr. fiir Hyg., XIV., 485, 1893. Tamamcbeff : Experiences sur les vaccins pbeniques de Haflkine. Ann. de I'lnst. Pasteur, 1892, p. 713. Van Ermengen ; Die Ferran'scben Impfungen. Deutscb. Med. Wcbscbr., 1885, No. 29. Vincenzi : Ueber intraperitoiiaale Einspritzung von Kocb'schen Kom- mabacillen bei Meerscbweiuschen. Deutscb. Med. Wcbscbr., 1887, Nos. 17 and 26. Wassermann, A. : Untersucbungen iiber Immunitat gegen Cbolera asiatica. Zeitscbr. fur Hyg.. XIV., 35, 1898. Westbrook, F. : Contribution a I'etude des toxiues du cbolera. Ann. de I'Inst. Pasteur, 1894, p. 318. Winter et Lesage : Contribution a I'etude du poison cholerique. Bull. Med., 1890, p. 328. Woodbead, G. S. : Some Notes on Haflfkine's Protective Inoculation against Cholera. Edinburgh Hospital Reports, 1893, p. 76. IV. DIPHTHERIA. Diphtheria is g-enerally recognized by physicians as a specific infectious disease, and, owing to its wide preva- lence and fatal character, a precise knowledge of its eti- ology is of the greatest importance. Until, as a result of recent researches, this was determined, pathologists were in doubt as to whether diphtheria should be considered as primarily a local infection, or whether the local mani- festations were secondary to a general systemic infec- tion. But this question appears now to be definitely settled in favor of the former view. We have to-day a very precise knowledge of the specific infecting agent, and have evidence that it produces during its growth a very potent toxic substance, the absorption of which from the seat of local infection accounts in a satisfactory manner for the general symptoms of the disease, which are due to toxaemia and not to an invasion of the blood and tissues by the pathogenic microorganism produc- ing it. Numerous researches by competent bacteriologists have failed to demonstrate the presence of bacteria in the blood of patients suffering from diphtheria, but various microorganisms have been obtained in cultures from diphtheritic pseudo-membranes, and may be demon- strated by the microscopical examination of stained prep- arations. Among these are the well-known pus organ- DIPHTHEEIA. 149 isms, and especially tlie streptococcus pyogenes, wliicli appears to be very commonly present, and is the active agent in the production of certain forms of pseudo-diph- theria. But the malig-nant, specific diphtheria, so well known in this country and in Europe, has been demon- strated by the recent researches of bacteriologists to be due to a bacillus, first recognized by Klebs in stained preparations of diphtheritic false membranes (1883), and cultivated and described by Lofiier in 1884. In his first publication Loffler did not claim to have fully demon- strated the etiological relation of this bacillus, but this appears to be fully established by subsequent researches. In his first research Lofiier studied twenty -five cases, and in the greater number of them found in stained preparations the bacillus previously described by Klebs. From six of these cases he obtained it in pure cultures, and by experiments on pigeons, chickens, rabbits, and guinea-pigs proved that it gave rise to a diphtheritic in- flammation when inoculated into the mucous membrane of the trachea, conjunctiva, pharynx, or vagina. In a second communication Lofiier reported his success in finding the same bacillus in ten additional cases, and also that he had isolated from the same source a nou -pathogenic ba- cillus which resembled it very closely. This pseudo- diphtheria bacillus has since been found by other bacte- riologists (Von Hoffman, Roux and Yersin), and it is uncertain whether it is to be considered a distinct spe- cies, or a non-pathogenic variety of the diphtheria bacil- lus as maintained by Eoux and Yersin. But its occasional presence does not invalidate the very positive experi- mental evidence relating to the specific pathogenic power of the true diphtheria bacillus. According to Roux and Yersin, " attenuated varieties " 150 IMMUNITY AND SERUM-THERAPY. of the diphtheria bacillus may be obtained by cultivating- it at a temperature of 39.5" to 40° C. in a current of air ; and these authors suggest that a similar attenuation of pathogenic power may occur in the fauces of convales- cents from the disease, and that possibly the similar non-pathogenic bacilli which have been described by various investigators have originated in this v^ay from the true diphtheria bacillus. These authors further state, in favor of this view, that from diphtheritic false mem- brane, preserved by them in a desiccated condition for five months, they obtained numerous colonies of the ba- cillus in question, but that the cultures were destitute of pathogenic virulence. They say : " It is then possible, by commencing with a virulent bacillus of diphtheria, to obtain artificially a bacillus with- out virulence, quite similar to the attenuated bacilli wdiich may be obtained from a benign diphtheritic an- gina, or even from the mouth of certain persons in good health. This microbe, obtained aiiificiallj^, resembles completely the pseudo-diphtheritic bacillus ; like it, it grows more abundantly at a low temperature ; it renders bouillon more rapidly alkaline ; it grows with difficulty in the absence of oxygen." Subcutaneous inoculations in guinea-pigs of a small quantity of a pure culture of the bacillus (0.1 to 0.5 c.c. of a bouillon culture) cause death in from one to four or five days. The usual changes observed at the autopsy are — " An extensive local oedema, with more or less hyper- emia and ecchymosis at the site of inoculation, fre- quently swollen and reddened lymphatic glands, in- creased serous fluid in the peritoneum, pleura, and pericardium, enlarged and hemorrhagic suprarenal cap- DIPHTHERIA. 151 sules, occasionally slightly swollen spleen, sometimes fatty degenerations in the liver, kidney, and myocardium. We have always found the Liiffler bacilli at the seat of inoculation most abundant in a grayish-white, librino- purulent exudate present at the point of inoculation, and becoming fewer at a distance from this, so that the more remote 'parts of the cedematous fluid do not contain any bacilli " (Welch and Abbott). The authors quoted agree with Liiffler and others in stating that the bacillus is only found at the point of in- oculation. In all cases their cultures from the blood and from the various organs gave a negative result. Rabbits are not so susceptible, and may recover after the subcutaneous inoculation of very small doses, but usually die in from four to twenty days when 2 to 4 c.c. of a bouillon culture have been introduced beneath the skin. In these animals, also, there is an extensive local cedema, enlargement of the neighboring lymphatic glands, and a fatty degeneration of the liver. Eoux and Yersin have shown that in rabbits, w^hen death does not ensue too quickly, paralysis of the posterior extremities frequently occurs, thus completing the experimental proof of the specific pathogenic power of pure cultures of this bacillus. Similar symptoms are produced in pigeons by the sub- cutaneous inoculation of 0.5 c.c. or more, but they com- monly recover when the quantity is reduced to 0.2 c.c. (Roux and Yersin), The rat and the mouse have a remarkable immunity from the eflfects of this poison. Thus, according to Roux and Yersin, a dose of 2 c.c, which would kill in sixty hours a rabbit weighing 3 kilogr., is without effect upon a mouse which weighs only 10 gm. 152 IMMUNITY AND SERUM-TIIERAPY. Old cultures are somewhat less yirulent than fresh ones, but when replanted in a fresh culture medium they manifest their original yirulcnce. Thus a culture upon blood-serum which was five months old was found by Roux and Yersin to kill a guinea-pig- in five days, but when replanted it killed a second animal of the same species in twenty-four hours. Evidently a microorganism which destroys the life of a susceptible animal when injected beneath its skin in small quantity, and which nevertheless is only found in the vicinity of the point of inoculation, must owe its pathogenic power to the formation of some potent toxic substance, which, being absorbed, gives rise to toxaemia and death. This inference in the case of the diphtheria bacillus is fully sustained by the results of recent exper- imental investigations. Eoux and Yersin (1888) first demonstrated the pathogenic power of cultures which had been filtered through porous porcelain. Old cultures were found by these experimenters to contain more of the toxic substance than recent ones, and to cause the death of a guinea-pig in a dose of 2 c.c. in less than twenty-four hours. The filtered cultures produced in these animals the same eflfects as those containing the bacilli — local cedema, hemorrhagic congestion of the or- gans, effusion into the pleural cavity. Somewhat larger doses were fatal to rabbits, and a few drops injected sub- cutaneously sufficed to kill a small bird within a few hours. In their second paper (1889) the authors men- tioned state that so long as the reaction of a culture in bouillon is acid, its toxic power is comparatively slight, but that in old cultures the reaction is alkaline, and in these the toxic potency is greatly augmented. With such a culture, filtered after having been kept for thirty days, DIPHTHEKIA. 153 a dose of one-eightli of a cubic centimetre injected sub- cutaneously, sufficed to kill a g-uinea-pig- ; and in larger amounts it proved to be fatal to dog-s when injected directly into the circulation through a vein. The same authors, in discussing the nature of the poi- son in their filtered cultures, infer that it is related to the diastases, and state that its toxic potency is very much reduced by exposure to a comparatively low tem- perature — 58° C, for two hours — and completely de- stroyed by the boiling- temperature — 100° C, for twenty minutes. It was found to be insoluble in alcohol, and the precipitate obtained by adding- alcohol to an old culture proved to contain the toxic substance. Loffler also has obtained, by adding- five volumes of alcohol to one of a pure culture, a white precipitate, soluble in water, which killed rabbits in the dose of 0.1 to 0.2 gva. when injected beneath the skin of these animals. It g-ave rise to a local oedema and necrosis of the skin in the vicinity of the point of inoculation, and to hyper?emia of the internal organs. This deadly foxhi appears to be an albuminoid substance, but its exact chemical composition has not yet been determined. Brieg-er and Frankel (1891) obtained results correspond- ing with those previously reported by Eoux and Yersin. Their researches showed that the toxic substance con- tained in diphtheria cultures is destroyed by a tempera- ture of 60° C. ; that it is soluble in water, and insoluble in alcohol ; that it does not pass through a dialyzing membrane, and has not the chemical characters of the ptomaines or toxins, but is an albuminous body — a toxal- bumin. It was obtained by the authors named b}^ pre- cipitation with slightly acidified (acetic acid) alcohol ; the precipitate, after being washed in a dialyzer and dried 154 I^IMUNITY AND SERUM-THERAPY. • in a vacuum at a temperature'of 40" C, was a snow-white, amorphous, crnmblin"- mass. Wasscrmann and Proskauer (1892) found that the alco- holic precipitate from diphtheria cultures contains two diiferent substances, which are disting-uished by their dif- ferent degrees of solubility in diluted and absolute alco- hol ; both, however, give the usual reactions of albumi- nous bodies, and pass very slowly through a dialyzing membrane. Only one of these substances laossesses toxic properties. After the removal of peptone and globulin from the filtered cultures, these were evaporated and a precipitate obtained of one of the albuminous substances by means of sixty per cent, to seventy per cent, alcohol. The other substance remained in solution, and was sub- sequently obtained by precipitation with absolute alco- hol. The substance first obtained by this method is toxic, and the other precipitate is not. The authors named succeeded in killing rabbits with the toxalbumin obtained in this way, but were not able to produce im- munity in these animals by the injection of non-fatal doses. Frankel (1891) had previously reported his faihn-e to immunize guinea-pigs by the injection of the dry pre- cipitate, obtained in his experiments from dii)htheria cultures ; but when filtered cultures, or cultures sterilized by heat (55" C. for one hour), were injected into these ani- mals, they showed an increased resistance to the patho- genic action of virulent cultures. Still better results were obtained when 10 c.c. of a bouillon culture, heated to 100° C, were injected subcutaneously, but still this method was not entirely reliable. But true immunity was established by injecting into the peritoneal cavity 10 to 20 c.c. of a bouillon culture heated to 65" to 70° C. for one hour. The immunity was not fully established until DIPHTHEEIA. 155 about fourteen days after the protective inoculation. Friinkel arrives at the conclusion that the cultures must contain an immunizing- substance as well as a toxic pro- teid, as the diphtheria toxalbumin is destroyed by the temperature (65° to 70° C.) used in the preparation of his cultures for producing- immunity. Behring-, in the same year (1891), commenced his exper- iments upon diphtheria immunity. Guinea-pigs were made immune by the use of sterilized cultures, and by inoculations with virulent cultures, four weeks old, to which iodine terchloride had been added in the propor- tion of 1 to 500^ the mixture was allowed to stand for sixteen hours. Animals were also immunized by inject- ing beneath the skin a virulent culture of the bacillus, and then treating them with subcutaneous injections of iodine terchloride (2 c.c), which was thrown under the skin for three days in succession in the vicinity of the point of inoculation. The guinea-pigs treated in this way remained sick for some time, but finally recovered and were subsequently immune. Still better results were obtained when rabbits were subjected to the same treatment. The animals were immune against the toxic action of sterilized cultures, as well as against infection by virulent diphtheria bacilli. In subsequent experiments (1892) Behring and Wer- nicke used cultures which had been attenuated by con- tact with iodine terchloride for from thirty-six to forty- eight hours, and proved that the method could be suc- cessfully employed in immunizing sheep ; and the fact was ascertained that blood-serum from an immune ani- mal could be used with success in arresting diphtheritic infection in susceptible animals. To preserve the serum, which they obtained from immunized sheep, rabbits, and 156 IMMUNITY AND SERUM-THERAPY. giiinea-pigs, they added to it 0.5 per cent, of pure carbolic acid. For producing- immunity tliey found that a smaller amount of serum was required than was necessary for the cure of an animal already infected. If the injection was made immediately after infection from one and a half to two times the amount was required ; eight hours after infection the amount was three times as great, and twenty- four to thirty-six hours after infection the dose required was eight times the immunizing dose. The immunizing value of blood-serum from different animals was estimated by finding the smallest dose which would protect an animal from fatal infection by the mini- mum lethal dose of a culture, the toxic potency of which had been carefully determined. The value is expressed in figures which give the proportion required comi:)ared with the body-weight of the animal. Thus an immunizing value of 100 would mean that 1 gm. of the serum is suffi- cient to protect an animal weighing 100 gm. from the fatal effect which would be produced in a control animal of the same weight by infection with a virulent culture of the diphtheria bacillus in the minimum dose required to pro- duce this result. The cultures employed are made in bouillon containing one per cent, of peptone ; they are inoculated from agar cultures and are kept in the incu- bating oven for two days. Cultures prepared in this way were found to be quite uniform in their pathogenic viru- lence as tested upon guinea-pigs. But when cultures are kept for some time there is an increase in virulence. Thus a culture obtained from a fatal case of diphtlieria which in 1890 killed guinea-pigs in three to four days, when injected subcutaneously in the dose of 0.1 c.c. (two days old bouillon culture), at the end of a year was fatal to these animals in the dose of 0.025 c.c. This increase DIPHTHERIA. 157 in virulence is ascribed to the fact that the cultures were renewed at long- intervals. More recently (1894) Behring has lixed a standard for what he calls a normal therapeutic serum. This is a serum which when injected into guinea-pig-s in the pro- portion of 1 to 5,000 of body-weight saves the animal from the fatal effects of ten times the minimum dose of a culture in bouillon, two days old, which would kill a control animal not treated. In a recent communication (November, 1894) Behring states his conclusion that for producing immunity in man, 150 normal antitoxin units should be given, instead of 60 as he had previously recommended. The serum manufactured under his direction is said (September, 1894) to be of two kinds — one, obtained from the horse, has a value of 60 normal antitoxin units ; the other has a value of 140 units. Of the weaker serum Behring says experience has demonstrated that for chil- dren under ten years of age 10 c.c. is sufficient to arrest the progress of the disease and effect a cure if given within two or three days from the outset of the attack. For producing immunity in children subject to infection, one-tenth of this amount (1 c.c.) is said to be sufficient. Of the stronger serum 1 c.c. is sufficient to arrest the disease during the incubation period ; and, according to Behring, out of 100 cases treated during- the first forty- eight hours with the single therapeutic dose (10 c.c. -of serum having a value of 60 = 600 normal units) not 5 will die. The later the treatment is commenced the larger will be the dose required. Behring further states that the diphtheria antitoxin has no injurious effect upon animals in the largest doses that have been employed, and that aside from its antitoxic power its iiroperties 158 IMMUNITY AND SERUM-THERAPY. are entirely negative so far as living animals are con- cerned. Aronson (1893), in experiments on dogs, succeeded in producing immunity by the use of attenuated cultures, or of cultures to which formaldehyd had been added ; also by feeding the animal large quantities of diphtheria bouillon; and, finally, by injection of the blood of natur- ally immune animals (white rats) into which large quan- tities (10 c.c.) of a virulent culture had been injected. Two months after receiving several such injections it was found that 0.2 gm. of blood-serum from the rat sufficed to save a guinea-i3ig from ftital infection. In experiments on dogs an immunity was established in six weeks by the injection of a large amount of a virulent culture, which had a value of 1 to 30,000, i.e., 0,01 c.c. of this serum sufficed to protect a guinea-pig weighing 300 gm. From 100 gm. of this serum Aronson claims to have ob- tained 0.8 gm. of a substance which had a value of 1 to 500,000, as tested in the treatment of an animal which had received ten times the minimum fatal dose of a two days' bouillon culture. A ten per cent, solution of this substance had, therefore, ten times the value of Behring's " normal-serum." The precipitated antitoxin was soluble in water, and more readily in a slightly alkaline solution, and gave all the reactions of an albuminous body. When dried in vacuo at 40° C, and then heated to 102° C, it still retained its antitoxic potency. Ehrlich, Kossel, and Wassermann (1894) have made ex- periments upon goats, which they found very suscep- tible to the action of the diphtheria poison. Sterilized cultures were first injected in gradually increasing amounts and later virulent cultures. In this way they obtained a serum which has a value sixtj' times that of DIPHTHERIA. 159 Behring's " normal serum." In a subsequent communi- cation (1894) Wassermann gives an account of his experi- ments with the milk of immunized goats, which contains the antitoxin in considerable quantity, and from which it was obtained in a concentrated form by the following method : The milk is obtained in sterilized vessels and 20 c.c. of normal hydrochloric acid is added to each litre ; a sufficient quantity of rennet is then added to coagulate the casein, and this is separated from the liquid, which is then shaken up with chloroform for some time. The liquid is now allowed to stand in order that the butter, which has been dissolved by the chloroform, may sink to the bottom. The clear liquid is then decanted and the antitoxin precipitated from it by means of ammonium sulphate (thirty to thirty-three per cent.). The precipi- tate is rapidly dried upon porous porcelain plates, in vacuo, and then dissolved in water in the proportion of 10 parts for 100 of milk first employed — a concentration to one-tenth. Of this solution 0.125 c.c. was found to neutralize 0.9 c.c. of a toxin which killed guinea-pigs, weighing 500 gm. in the dose of 0.1 c.c. This toxin was an old bouillon culture of the diphtheria bacillus to which 0.5 per cent, of carbolic acid had been added to preserve it. In a communication of the same date EhrKch and Wassermann report that they have for some time had a cow immunized to such a degree that 1 c.c. of its milk protects guinea-pigs from the fatal effects of 0.9 c.c. of the above-mentioned toxin. The antitoxic value of the milk of an immunized cow or goat, as compared with that of its blood, is estimated by Ehrlich and Wassermann as from 1 to 15 to 1 to 30— usually about 1 to 20. Aronson, in testing his antitoxin, uses a bouillon cult- ure of the diphtheria bacillus two and one-half mouths 160 IMMUNITY AND SERUM-THERAPY. old, which he preserves by the addition of 0.3 per cent, of trikresol. He fiuds that the immunity which results from injections of the antitoxin is established at once ; that it is not accompanied by any reaction or symptom of sickness ; and that it is of comparatively short dura- tion. As a result of extended experiments made at the Pas- teur Institute in Paris, Koux has perfected the following method for the production of an antitoxin suitable for use in the treatment of diphtheria in man. The horse has been found the most suitable animal for this pur- pose, on account of his slight susceptibility and the ease with which a high degree of immunity can be estab- lished ; and because of the large amount of blood that may be drawn without injury to the animal. Roux pre- pares his toxin by cultivating the diphtheria bacillus in a slightly alkaline bouillon made from beef and contain- ing two per cent, of jieptone and 0.5 per cent, of sodium chloride. This medium is placed in flat-bottomed flasks, and should not be more than half an inch in depth. Two glass-tubes pass into the flask, which serve for inlet and outlet tubes to be used in passing a current of air over the cultures. This is commenced when the growth is fairly started, at the end of twenty-four hours, and the air should be moist to prevent the evaporation of the cult- ure. In Roux's laboratory a flask is used which has a tube attached to one side, about an inch from the bottom, and which is known as a Fernbach flask. A flocculent deposit falls to the bottom and gradually accumulates for about a month. This consists of bacilli which have for the most part lost their vitality and are tindergoing degeneration. At th^ end of thirty days, during which time they are kept in an incubating oven at a tempera- DIPHTHERIA. 161 ture of 37° C, tlie cultures are passed through a Pasteur- Chamberland lilter, and 0.5 per cent, of carbolic acid may be added in order to preserve them. This filtrate is so toxic that a dose of 0.1 c.c. will kill a g-uinea-pig- weigh- ing 500 gm. in less than forty-eight hours. A healthy horse is selected and receives at first a dose of 0.5 c.c. of the filtered culture (or of the clear fluid obtained from a culture by decautation and containing 0.5 per cent, of carbolic acid). The dose is gradually increased at inter- vals of a few days, and is followed each time by some febrile reaction and tumefaction at the point of inocula- tion. When the reaction is excessive, a little Gram's solution is added to the following dose. The usual plan of treatment is stated by Kinyoun as follows : "First day, 1 to 2 c.c. of j-mre toxins, of which 1 to 10 c.c. fatal to a 500-gm. guinea-pig; eighth day, 1 c.c; fourteenth day, Ig c.c. ; twentieth daj", 2 c.c. ; twenty— eighth da}' , 3 c.c. ; thirty-third day, 5 c.5. ; tliirt3^-eighth day, 8 c.c. ; forty -third day, 10 c.c. ; forty-seventh day, 20 c.c. ; fifty -first day, 30 c.c. ; fifty-sixth day, 50 c.c. ; sixty- second day, 50 c.c. ; sixty-eighth day, 60 c.c. ; seventy- fourth day, 100 c.c. ; eightieth day, 250 c.c. ; eighty-eighth day, 250 c.c. " When the first injections are given there is quite a marked local and general reaction to the poison ; there is an oedema at the point of the injection, which is followed by a distinct inflammatory process — hard in the centre and soft and oedematous at its periphery. The general reaction is manifested by a rise in the temperature, 1° to 2° C, loss of appetite, and occasionally cramps: The re- action must be taken as the guide in the future dosage, and a sufficient time must be allowed to elapse between the injections for the complete recovery from the general and local effects. As the quantity of the toxins is in- creased the general effects generally decrease, perhaps a 11 162 IMMUNITY AND SERUM-TIIEnAPY. rise of a degree for twenty -four hours. The local effect partakes more of an oedema, and has the character of an intiammation. " At a certain stage, usually after two months' treat- ment, when 50 to 60 c.c. can be injected without harm, there is no general reaction, but a large oedema at the site of the injection, which disapjjears within from tAvent}-- four to forty-eight hours. Toward the last, even when 200 to 300 c.c. are given, there is only an enormous oede- ma, which disappears within from twelve to eighteen hours. When these inordinateh' large quantities can be given with only a local reaction being manifest, the horse has come well under the influence, and the blood will be found to be rich in the antitoxin. " There is a curious fact well worth noting : At the end of the second month of the treatment, when the horse can bear as much as 50 to 60 c.c. of the toxins without discom- fort, the blood will be found to contain but little of the antitoxin. The antitoxin only appears after repeated stimulation of the cells (?) by the large and frequent doses of the toxijis." The subcutaneous injections do not jdeld a serum as rich in the antitoxins as when the toxins are injected di- rectly into the blood-current. '\Mien it is desired to do this, toward the last of the treatment the toxins are in- jected directl}^ into the jugular vein. The process is tedious and requires a longer time, and for practical pur- poses has not been found so satisfactory as the simple subcutaneous injection! The strength of the serum is tested by using j^oung guinea-pigs of 500 gms. weight. One gr. of the serum usually will protect 50,000 gms. of guinea-pig against a fresh virulent culture of the Bacil- lus dipJitherlae. This is the strength that is used in the hospitals. By the intravenous injections a serum of the protective strength of 1 to 100,000 can be obtained. DIPHTHERIA. 163 When fully immunized from six to eight litres of blood may be taken from a horse at one time, but as a rule it is better not to take more than three. The blood is drawn from the jugular vein, by means of a small trocar and cannula, into wide-mouthed bottles having a capacity of 21 litres ; these are jalaced in an ice-chest for twenty- four hours to give time for the separation of the serum, which is then transferred to smaller receptacles for pres- ervation. The dose of serum prepared in this way, when used to protect from diphtheria infection, is 5 c.c. for a child uq- der ten years of age, and 10 c.c. for older children. This does not afford an absolute protection, but is believed to be generally effective, and in case of failure the attack is said to be of a mild character. The curative dose of Koux's serum is 20 c.c. for children, and 30 to 40 c.c. for l)atients over fifteen years of age. The larger dose is divided and given, at the same time, by subcutaneous in- jection in two places. Antisejitic precautions are taken in giving these injections, and a little absorbent cotton is placed over the puncture. Hesults of Treatment with Serum of Immune Animals. We take the following statistics from a recent (Decem- ber, 1894) paper by Woodhead : " It may be pointed out from statistics that no fewer than 13,694 cases were notified in London during the year 1893. Among these cases there was a mortality of 3,195 or 23.3 per cent, (see The Lancet corrected statis- tics). In a series of most carefully prepared and tabu- lated statistics, those of the Metropolitan Asylums Board's hospitals which receive diphtheria patients 164 IMMUNITY AXD SEKUM-THERAPY. Table I. — Metropolitan Asylums Board : Admisgions and Case Mor- tality, Diphtheria, 1888-93. Year. No. of Admissions. No. of Deaths. Percentage of Case Mortality. 1888 99 723 942 1312 2009 2848 46 275 316 397 583 865 46 4 1889 38 1890 . 1891 33.5 30 2 1892 29 1893 30 3 Note. — Diptitlieria cases have only been admitted into the hospitals since October 23, 18b8. Table II. — Shairing the Mortality at Various Ages from Diphtheria admiiled into the Metropolitan Asylums Board's Ho&pitali in the Years 1888-93. Ages. Cases Admitted. Died. Mortality per cent. Under 1 146 447 039 826 913 102 291 388 416 400 69.9 1 to2 65.1 2to3 60.7 3 to 4 50.4 4 to 5 43.8 Totals under 5 2,971 1,597 53.0 5 to 10 2,402 885 588 420 209 137 87 50 27 13 705 93 23 22 28.6 10 to 15 15 to 20 10.5 3.9 20 to 25 5.2 25 to 30 30 to 35 35 to 40 13 5 5 6 4 4 1 3 4.8 3.6 5.7 1 40 to 45 45 to 50 50 to 55 ;- 16.8 1 55 to 60 11 6 And upward J Totals 7,932 2,481 31.3 DIPHTHERIA. Table III. — Serum Treatment of Diphtheria. 165 u 1" Percentage of Mor- tality. Percentage of Previous Mortality. Rou.K, Marti ii, ) and Cbaillou. \ Ehrligb, Kossel, \ and Wasser- > maun ) * Canon Paris 448 220 15 34 60 89 233 121 35 96 128 255 19 70 65 2 32 79 109 52 3 6 19 12 54 40 5 37 17 31 4 7 18 8 9 24.5 23.6 20.0 18.0 33.3 12.3 23.0 33.1 14.2 38.5 13.2 12.1 21.0 10.0 28.8 ..IT 25.0 11.4 51. 7^ 34. 7§ 25 Berlin *Scbubert 1. *yoswinkel Ebrlich (1 46.5 flvQssel *Korte .' '< 53 8 Bokai Heubner ^ . . Katz Aronsou Budapest Beriin ".'.*.'.".;!'.; 53.8 62.5 38.911 32 5-41 7 Ranke and Oertel. The Lancet Weibgen Muchleck Dr. White Cases reported in British Medical Austrian Riviera Berlin Pbiladelphia i WillVl Parker ) - H s p i t a 1 , [- ( New York. . . ) The Lancet and ] Journal f 40.0 42.7 * Canon, Schubert, and Voswinkel's statistics, and probably also the bulk of Korte's, are really included in the Ehrlich, Kossel, and Wassermann's table. + Kossel's statistics appear to be simply an extension of those given by Ehr- lich, Kossel, and Wassermann. I Mortalitj' in Trousseau Hospital at same period without use of serum 63.2 per cent. § Based on previous seven years' statistics. 1 Based on three years' statistics. Last year's mortality was 41.7 per cent. 1[ Tvyo not treated died. (Table I.) we find that during- the year 1893 the case mor- tality among- eases of diphtheria admitted to the Avards of these hospitals was a little OAer thirt}' per cent. — under conditions which we may say are practically unknown in France and in most parts of Germany and Austria. This is sufficiently startling-, but if we examine earlier statis- 166 IMMUNITY AND SERUM-THERAPY. tics we find that duriner tlie years 1888-90 the case mor- tality, as indicated by tiie admissions and deaths, "svas much higher. On reference to Table I. it will be seen that since the year 1888 the case death-rate has fallen from 4G.4 to 30.3 per cent., althoug-h the number of cases of diphtheria treated has risen enormously. The mortal- ity from diphtheria in London during- the same period has also risen very considerably. The case mortality at different ages is brought out on reference to Table II. Comparing these results of treatment in our hospitals with those obtained in the Paris and Berlin hospitals (see Table III.), the records of which are published,. it is at once seen how much more favorable are the results .ob- tained in the hospitals of the Metropolitan As^dums Board. It will be noted, however, that the figures apply- ing to the earlier years represented a case mortality al- most as high as obtained in the French and German hos- pitals immediately before the antitoxic-serum treatment was introduced. The mortality in hospitals must always be greater than in private practice from the fact that so many of the cases are so serious ; some of the patients, indeed, are actually moribund when aelmitted." Fischer (January, 1895) reports 34 cases — 30 malignant and 4 mild — treated with a mortality of two = 5.8 per cent. He says: " These cases were not selected, for some were poorly nourished, some in excellent vitality, with careful nurs- ing and good hj' giene. The main point was to appl}' the antitoxin as early as possible, and counteract the septic matter absorbed, and thereby avoid complications, be- sides using the local treatment of swabbing the throat with a 1 to 2,000' bichloride of mercury solution, using a fresh swab for each application, and burning the same immediately after using it. "The technique of injection is simple. Having prop- erly sterilized the syringe by boiling and using 0.5 per DIPHTHERIA. 167 cent, tricresol, I commence by injecting- 10 c.c. in mild cases, and 20 c.c. in malignant cases, by i^incliing a fold of the skin in tlie intrascapular region, and allowing the serum to be slowly injected. I believe it proper, however, to have a syringe of suitable size and inject the required amount, rather than inject several places. The calibre of the latter must necessarily be quite large, owing to the thickness of the serum, which is at times rather mucilag- inous. It is proper to note all differences and effects on the false membrane and the swelling of the glands, the behavior of the temi:>erature, the condition of the urine, the effect on the heart, and especially the pulse. " There should be no hesitation in injecting on the sec- ond day, and, if no effect is seen, repeating the injection on the third day, as there is absolutely no risk from the injection. It is a perfectly safe remedy, and shows no immediate reaction. It differs from tuberculin and vac- cine in that it causes no reaction. A case of antitoxin treatment will show no symptoms directly attributable to the remedy, unless it be in some cases of urticaria. The temperature does not fall by crisis, but by lysis, with an- titoxin treatment. Massage of the serum after the injec- tion should not be practised, according to Heubner, Aronson, Baginsky, and others." The efficacy of the antitoxin treatment has been com- batted in Berlin by Hansemann and others. The oppo- nents of the treatment point to a considerable number of recorded cases in which relapses have occurred after ap- parent recovery following treatment with the antiseptic serum ; also to the occurrence of diphtheria in children who had been treated with immunizing doses of the serum ; and to the failure to cure in a considerable num- ber of cases notwithstanding the administration of large doses of the antitoxic serum. All of these facts must be admitted, but the experimental (on animals) and clinical 168 IMMIXITY AND SEIIUM-THEIIAPY. evidence heretofore submitted appears to establish the value of the treatment when applied before the disease has i^rogressed too far. It must be remembered that the antitoxin has no power to destroy the diphtheria bacilli, or to relieve the suffocation resulting from obstruction of the larynx, or to cure an acute parenchymatous nephritis due to the action of the deadly toxin elaborated by the Klebs-Loffler bacillus. Saltmann (1895) reports a mortality of 39.8 per cent, in 71 cas3S treated in the children's hospital at Leipsic be- fore the use of the antitoxin. The report relates to cases treated from April 1, to December 31, 1894. During the last five months of this period 122 cases were treated and most of them received the antitoxin. The mortality was 18 per cent. In an article contributed to McClure^s Magazine for March, 1895, Dr. Hermann Big-g-s, of New York, saj-s : " That prepared in this country under the supervision of the New York City Health Department has, at the time of writing, been already employed in more than one hun- dred and fifty cases, and the mortalit}^ in the cases thus treated has been about twelve per cent." Klebs has proposed to treat diphtheria with a prepa- ration obtained from cultures of the diphtheria bacillus which he calls antidiptherin. The clinical experiments made by Yulpius and by Zappert show that the treat- ment has no value and it has been abandoned. BIBLIOGRAPHY. Abel, R. : Ueber die Scluitzkraft dcs Blutserums von Diplitberierekon- valescenten, etc. Dtscbe Med. AVcbsolir. , 1894. p. 899. Aronson : Experimeiitale Untcrsucbunsren iiljcr Dipbtberie und die im- munisierende Substanzdes Blutserums. Bed. klin. Wcbscbr., 1893, Nos. 25 and 26 ; Centialbl. f. Bakteriol., XIV., p. 260. DIPnTHERIA. 169 Aronson : Ueber Diplithcrieantitoxin losungzii Immunisierungszweck- en. Dtscb. med. Wchscbv., 1894, p. 431. Zur Diphtberiebeihiugsfiage. Dtscb. Med. Wcbscbr., 1894, p. 384. Weitere Untersucbungeu iibcr Dipbtberie und das Dipbtberie- autitoxiti. Berl. kUn. Wocbeuscbr., 1894, pp. 356, 42.1, 453. Abst. in Ceutralbl., XV., 926. Meine Stelluug in der Dipbtberieantitoxiufrage. Berl. klin. Wcbscbr., 1894. No. 47, p. 1077. Iiiimunisieruugs- uud Heilversucbe bei der Diplitberie mittelst Antitoxin. Wien med. Wcbscbr., 1894, Nos. 46-48, pp. 1953, 2001, 2046. Bebring : Untersucbnngen iiber das Zustandekommen der Dipbtberie- immunitiit bei Tbieren. Deutscbe Med. Wcbscbr., 1890, No. 50. Die Blutserumtberapie bei Dipbtberie und Tetanus. Zeitscbr. f. Hygiene, XII., 1, 1883. Zur Bebandluug der Dipbtlierie mit Dipbtberiebeilserum. Dtscbe. med. Wcbscbr.. 1893, pp. 543, 594, 623, 637. Weitere Bemerkungen zur Dipblberiebeilungst'rage. Dtscbe. Med. Wcbscbr., 1894, No. 32, p. 645. Zur Diplitlierieimmunisierungsfrage. Dtscbe. med. Wcbscbr. 1894, No. 46, p. 865. Die Blutserumtberapie zur Dipbtberiebebandlung des Menscben. Berl. klin. Wcbscbr., 1894, p. 827. Bebring und Kitasato : Ueber das Zustandekommen der Dipbtberieim- munitiit und der Tetanusimmunitat bei Tbieren. Deutscbe med. Wcbscbr., 1890, No. 49. Bebring und Wernicke : Ueber Immunisierung und Heilung von Ver- sucbstbieren bei der Dipbtberie. Zeitscbr. f. Hygiene, XII., 10, 1892. Bebring, Boer, und Kossel : Zur Bebandlung dipbtberiekranker Men- scben mit Dipbtberiebeilserum. Dtscbe. med. Wcbscbr., 1893, 389, 415. Bebring und Boer : Ueber die quantitative Bestimmung von Dipbtberie- antitoxin. Dtscbe. med. Wcbscbr.. 1894, No. 21. Bokai, J. : Mein auf der Dipbtberieabtbeilung des Stefaniekinder- spitals mit dem Behring'scben Heilserum bisber erzielten Resultnte. Pester Med. Cbirug. Presse., 1894, Nos. 44 and 45, pp. 1120, 1143. Brieger und Frankel : Untersucbungen iiber Bakteriengifte. Berl. klin. Wcbscbr., 1890, Nos. 11 and 12. Ueber Immuni-sierungsversucbe bei Dipbtberie. Berl. klin. Wcbscbr., 1890, No. 49. Canon : Zur Dipbtberiebebandlung mit Heilserum. Dtscli. med. Wcbscbr., 1894, Nos. 23 and 24. Ebrllcli und Kossel : Ueber die Anwendung des Diphtberieantitoxins. Zeitscbr. fiir Hygiene, Bd. XVII., 1894, p. 486. 170 IMMUNITY AND SERUM-THERAPY. Eliiiicb, P., u. Wassermann, A. : Ueberdie Gewinimng der Diplitherie- autiloxineaus Blutscrum uiid Milch iminunisierter Tliiore. Ztsclir. fiir Hygiene, 1894, Bd. XVIII., p. 238. EUrlich, Ivossel, und "Wassermann : Ueber Gewinnung uud Verwen- dung des Diphlberiebeilserums. Dtscb. med. Wocbenscbr., 1894, Xo.^G. Hausemauu : Mittheilungen iiber Diplitberie und das Dipbtheiie-Heil- serum. Berl. klin. Wcbscbr., 1894. p. 595. Hilbert, P. : Die Resultate der in der Kgl. mediziniscbeu Uuiversitats Poliklinik zu Kouigsberg ausgefiibrten Scbutz-und Heilimpfungen bei Dipbtherie. Berl. klin. Wcbscbr., 1894, Xo. 48, p. 1084. Kiuyon : The Seruuitberapy of Dipbtberia. Abst. of Sanitary lie- ports, vol. ix., Xo. 51, 1894. Klebs : Ein Beitrag zur Antidipbtbcrin Bebandlung. Dtscb. med. Wcbscbr., 1894, Xo. 18. Klemensiewicz, R., und Escbericb, Tb. : Ueber einen Scbutzkorper im Blute von Dipbtberie gebeilten Mensclieu. Centralbl. f. Bakteriol., XIII., 153, 1893. Korte, W.: Bericbt iiber die Bebandhing von 121 Dipbtberiekranken mit Bebring'scben Ileilserum im stadtiscben Krankenbaus am Ur- ban. Berl. klin. W(discbr., 1894, Xo. 46, p. 1039. Kossel, H. : Ueberdie Bebaudhing der Dipbtberie des Menscben mit Diptberiebeilserum. Zeitscbr. fur Hyg., XVII., 489, 1894. Koudrevetsky, B. : Kecbercbes e.xperimeutales sur I'immunisation contre la dipbtberie. Arcb. de ]VIed. experiui., 1893, p. 620. Kuprianow, J. : Experiinentelle Beitrage zur Frage der Immunilat bei Dipbtberie. Centralbl. f. Bakteriol., XVI.. 415, 1894. Lubinski, W. : Ueber eine Xachwirkung des Antitoxins bei Beband- lung der Dipbtberie. Dtscbe. med. Wcbscbr., 1894, Xo. 45, p. 857. Roux et Yersin : Contribution a I'etude de la dipbtberie. Ann. de rinst. Pasteur, t. ii., 1888, p. 629. Roux, E., Martin, L., et Cbaillou : Trois cents cas de dipbtberie traites par le serum antidipbtberique. Ann. de I'lnst. Pasteur, 1898, p. 640. Risso, A.: Ueber die Immunisation von Tbieren gegen Dipbtberitis und iiber die Serumiberapie. Abst. in Centralbl. fur Bakteriol., XVI. , 244, 1894. vSaUmann : Die Serum Bebandlung der Dipbtberie. Deutsche med. Wcbscbr., 1895, p. 53. Schubert, E. : Ueber die mit dem Bebring-Elirlicb'scben Diptberiebeil- .serum gemachten Erfabrungen. Dtscbe. med. Wcljscbr., 1894, p. 476. Smirnow : Ueber die Bebandlung der Dipbtberie mit Antitoxinen, die ohne Yermittclung des thieriscben Organismus darstellbar sind. Berl. klin. Wcbnscbr., 1894, p. 683 ; Centralbl., XVI., 546. DIPHTHERIA. 171 Strauss : Sur la serumtherapie. Rapport d'uue commission. Bull, de I'Acad. de Med., 1894, No. 43, p. 329. Uschinski, N. : Ueher die chemische Beschaffenheit des Diplitherie-nnd Choleragiftes. Bolnitscli. Gaz. Botkiua, 1893, pp. 437, 453, 485. (Russiau.) Vo.swinckel : Resultate der Heilserumtlierapie bei Diplitlierie. Abst. in Ceutralbl. f. Bakteriol., XVI., 548, 1894. Vulpius : Kritisclie Bemerkungen und praktische Erfalmngen iiber das Antidipbtherin Klebs. Dtscb. med. Wcbscbr. 1894, No. 6. Wassermann, A.: Ueber Konsentrieruiig der Dipbtberieantitoxin aus der Milcb immunisierter Tbiere. Ztscbr. f. Hygiene, 1894, Bd. XVIII., p. 235. Wernicke : Ein experimeuteller Beitrag zur Kenutniss des Loffler'scben Dipbtberiebacillus und zur " Blutserumtberapie." Arcb. f. Hyg., 1893, p. 193. Beitrag zur Kenntniss des Loifler'scben Dipbtberiebacillus und zur Blutserumtberapie bei Dipbtberie. Abst. in Centralbl. I'iir Bakteriol., XV., 898, 1894. Woodbead, G. S. : Tbe Diagnosis and Antitoxic Serum Treatment of Dipbtberia. Tbe Lancet, London, December, 15, 1894, p, 1409. Zappert, J.; Ueber die Heilwirkung des Antidipbtberin (Klebs). Wieu med. Wcbscbr., 1894, pp. 550, 616, 666, 708, 756. Zimmer E. : Untersucbungen iiber das Zustandekommeu der Dipbtberie- immuuitat. Dtscbe. med. Wcbscbr., 1893, No. 16. V. FOOT-AND-MOUTH DISEASE. This is an infectious disease of cattle, sheep, goats, and swine, the etiology of which, so far as the specific infec- tious agent is concerned, has not been determined. Schottelius has (1892) described a microorganism found by him in the contents of the vesicles of foot-and-mouth disease which seems to be peculiar to this disease, but he was not able to demonstrate its etiological relation by inoculation experiments. The microorganism referred to is a streptococcus so far as its morphology is con- cerned, but differs from the previously known strepto- cocci in being extremely motile. The extent to which the disease in question prevails in some parts of Europe is shown by the statistics for 1891 of the prevalence of this disease in Germany. According to the Reichsseuchenbericht it prevailed most exten- sively in the southern portion of Germany. The total number of infected farms was 47,865 ; the total number of infected cattle was 394,640 ; of sheep, 240,904 ; of goats, 3,378 ; of swnie, 182,208. Behla (1892) has made inocula- tion experiments with the filtered saliva of infected cattle to which he added one to two per cent, of carbolic acid, and claims to have produced immunity in young pigs and lambs. The duration of immunity is not, however, very long even in animals which have recovered from an attack of the disease — said to be from six months to three FOOT-AND-MOUTH DISEASE. 173 years — and a practical method of restricting" the disease by means of protective inoculations has not as yet been introduced. BIBLIOGEAPHY. Behla, R. : Zur Schutzimpfung bei Klauen- uud Maulseuchc. Beil. tierarztl. Wclisclir., 1892, No. 49. Sanfelice, F. : Ueber einen Befuiid an von Maul- und Klauenseiiclie befalleuen Tieren. Centralbl. f. Bakteriol., 1894. No. 23, p. 896. Schiitz : Impfversuche zum Schutze gegeu die Maul- und Klauenseuche. Arch, fiir Wissenscb. u. Prakt. Tierbeilk. 1894, No. 1, p. 1. Scbottelius : Ueber einen bakteriologiscben Befund bei Maul, und Kll,uenseuche. Centralbl., f. Bakteriol. Bd., XI., 1893, p. 566. VI. Glanders. The etiological relation of the bacillus discovered by Loffler and Schiitz, in 1882, to glanders is well estab- lished. Experiments by the bacteriologists named, and frequently repeated by others, show that pure cultiires of this bacillus injected into horses, asses, and other suscep- tible animals, produce genuine glanders. The field- mouse and the guinea-pig are especially susceptible to infection by experimental inoculations ; the cat and the goat may also be infected ; rabbits, sheep, and dogs are but slightly susceptible, and swine, cattle, white mice, and house-mice are immune. The toxic substances produced in cultures of the glan- ders bacillus when concentrated in the form of a glj^ceriu extract constitute the so-called mallein, which has been extensively used in the diagnosis of glanders in horses. As is the case when animals infected with tuberculosis are inoculated with tuberculin, animals infected with glanders have a decided rise of temperature after receiv- ing a sufficient dose of mallein beneath the skin. Babes (1892) reports that the toxic substance in cult- ures of the glanders bacillus may be obtained by precipi- tation with alcohol ; and that mallein obtained from filtered cultures to which glycerin has been added, or the alcoholic precipitate, may be successfully used for pro- tecting susceptible animals against glanders infection or GLANDERS. 175 for curing- the disease after infection. He has demon- strated the therapeutic value upon g-uiuea-pigs and upon two horses which are said to have been cured of chronic glanders. When large and repeated doses are injected into healthy animals they produce nephritis and general marasmus. The action upon horses infected with glanders is very marked and small doses may even cause death. Kresling (1892) recommends potato cultures as prefer- able to bouillon cultures for the preparation of mallein. The potatoes are to be washed, before sterilization, in a five per cent, bicarbonate of soda solution, "until the wash-water remains clear." They are then cooked for an hour and twenty minutes. After planting uj)on the sur- face glanders bacilli from a previous culture they are placed in an incubator at 36° to 36.5° C, with provision to l^revent them from becoming dry. At the end of two weeks the growth is removed with a platinum spatula and added to nine parts of water, in which it is well mixed by rubbing. It is then allowed to stand for twenty-four hours, after which it is sterilized for fifteen minutes at 110'' C. (a lower temperature would no doubt answer quite as well). After cooling it is passed through a Chamberlain filter by means of a pressure of six atmos- pheres. The filtrate is then carefully evaj)orated over a water-bath to one- fourth its volume, and to this concen- trated extract glycerin is added in the proportion of one part to two. The mixture is again sterilized in the auto- clave at 110° C. When injected into healthy horses in the dose of 2 c.c. this mallein does not cause an elevation of temperature exceeding 0.5° to 0.8" C. But 1 c.c. in- jected into a horse having glanders causes its tempera- ture to mount to 40" C, and at the point of inoculation a 176 IMMUNITY AND SEIiUM-TIIERAPY considerable swelling- is developed, which lasts from four to six days — in healthy horses a swelling- the size of a man's list is developed at the point of inoculation, which disappears within twenty-four hours. In Pasteur's laboratory, according- to Nocard (1892), mallein is prepared as follows : The g-landers bacillus is first made so virulent by successive inoculations in sus- ceptible animals that it will kill a rabbit or a white mouse in a few hours. This virulent bacillus is culti- vated in g-lycerin-pepton-flesh-infusion (five per cent, of glycerin and five per cent, of pei3ton). The cultures are kept in the inciibating- oven for four weeks at a tempera- ture of 31° C, and then sterilized in the autoclave at 110° C. They are then filtered throug-h paper and evaporated, in vacuo, over sulphuric acid, at a low temperature, to one-tenth of the original volume. The result is a sirup- like, dark brown, strong-smelling- liquid, which is about one-half glycerin. This can be preserved in a cool and dark place for a long time. When it is to be used nine parts of a 0.5 per cent, solution of carbolic acid are added to one part of the glycerin extract. The concentrated extract, when injected into a healthy horse in the dose of 0.5 to 1 c.c, causes a local swelling which disappears after two or three days. The temperature of the body is elevated from 1.5° to 2° C. as a result of the injection, and there is chilliness, loss of appetite, and debility. When the diluted mallein is injected in healthy animals in the dose of 2.5 c.c. no reaction occurs. On the other hand, this dose causes an intense febrile reaction in horses with glanders. There is a chill followed by an elevation of temperature amounting to 2° to 3° C, accompanied by dyspnoea and great debility ; in some cases the animal died as a result of the inoculation. GLANDERS. 177 For the preparation of the active substance in a dry condition, Foth gives the following directions : The cult- ures are evaporated at a temperature not exceeding- 80° C. to one-tenth of their volume, and filtered. The clear and thick, dark brown liquid is then slowly dropped into absolute alcohol (25 to 30 parts) with constant stirring. A flaky, white precipitate is thrown down, and accumu- lates as a i^ale yellow mass upon the sides and bottom of the vessel. After standing for twenty-four hours the alcohol is carefully drawn off and the i^recipitate washed with absolute alcohol. This is to be carefully done, and to avoid loss will require several days. The precipitate is then placed upon a thick paper filter and thoroughly washed by drawing alcohol through it by means of an exhaustion apparatus, after which the purified precipi- tate is collected and dried with care at a low temperature — best in a vacuum over sulphuric acid. A spongy, crum- bling mass is thus obtained, which is easily crushed to form an extremely light white powder. This is readily soluble in water. It is not at all hygroscopic, and can be preserved in a dry condition without difficulty. The dose for a horse is 0.1 gm. De Schweinitz and Kilborne, in a paper published in November, 1892, state that in December, 1890, they " Extracted from culture liquids of the bacillus mal- leus an albumose which appeared to be the active princi- ple in these cultures. At that time a preliminary exper- iment was conducted to see if this substance could be used to make guinea-pigs immune to the disease — glan- ders. The result was that out of a set of five, three vac- cinated and two checks, only one, a vaccinated animal, recovered from an inoculation of a glanders culture. This experiment has since been repeated with sets of ten 12 178 IMMUNITY AND SERUM-THERAPY. and twelve guinea-pigs each, with, at present writing, only negative results. A note of this work was published in the ' Annual lieport of the Department of Agriculture for 1891.' The albumose was best obtained from the cult- ures, after the removal of the germ, by means of a Pasteur filter, by precipitation with absolute alcohol, resolution in water, and reprecipitation." Babes (1892) claims to have succeeded in immunizing guinea-pigs against glanders by means of the toxic sub- stances contained in cultures of the bacillus. In a recent paper (1891) Foth has reported the results of extended experiments which have been made with his " Malleinum siccum " in Austro-Hungary. These results are stated as follows : The experiments were for the most part made by Pro- fessor Schiudelka, of Vienna. The tests were made with doses ranging from 0.01 gm. to 0.02 gm. The number of horses treated, for diagnostic purposes, was 455 ; of these 147 were examined post-mortem. In general the infected horses reacted and the others did not. A reaction of 2° C. and upward, running a typical course, was evidence that the animal was infected, and such animals were killed and carefully examined by autopsy. A reaction of 1.3° to 1.9° C, running a typical course, was taken as evidence that the animal was probably in- fected, and called for its isolation and a subsequent in- oculation after an interval of four weeks. A reaction of less than 1.2° C, or an atj'pical course of the febrile reaction, was taken as evidence of non-infection. The typical febrile reaction consisted in a rapid or gradual elevation, according to the dose, then a fall of some tenths of a degree, a subsequent elevation to the highest previously reached point or above, and a gradual GLANDERS. 179 fall to the normal. Tlie at3'pical reaction, which some- times occurs in healthy animals, consists in an early and rapid elevation followed by an equally rapid fall to the normal. To properly distinguish the typical temper- ature curve, upon which the diagnosis depends, hourly observations are considered necessary. Schiitz (1894^, as a result of his experiments on fifty- four horses, arrives at the conclusion that mallein may give rise to the so-called " typical reaction " in horses which are not infected with glanders. Hutyra and Preiz (1894), as a result of their extended researches, arrive at the conclusion that the use of mallein constitutes the most important means for the early diag- nosis of glanders in horses. They conclude that a temper- ature of 39.4° C. may be accepted as a safe positive mal- lein reaction. According to them the reaction commences from four to six hours after the injection, and reaches its maximum in from eight to fourteen hours — rarely in six- teen to twenty hours. The return to the normal occurs in from twenty-four to thii"ty-six hours. The authors last named give the following directions for the preparation of mallein : The virulence of the glanders bacillus is first increased by passing it through a series of guinea-pigs. Cultures are then made upon sterilized potato. When the culture and potato have become quite dry and dark colored they are collected in a glass dish and covered with a liquid consisting of equal parts of glycerin and dis- tilled water, containing 3 to 5 parts per thousand of mer- curic chloride. After standing for from ten to fourteen days in an incubating oven at 37.5° C, the liquid is fil- tered through paper and sterilized for an hour in a steam- sterilizer. This liquid remains sterile on account of the presence of mercuric chloride, and may be preserved a 180 IMMUNITY AND SERUM-THERAPY. long time without losing its activity. The dose is from 0.3 to 0.5 c.c, which is diluted to 3 c.c. with carbolic acid water (0.5 per cent, solution). The diluted solution may also be kept a long time without losing its activity. Bonome and Yivaldi (1892) have tested the action of mallein obtained by precipitation with alcohol upon various animals. Guinea-pig^ were found to resist com- paratively large doses (10 to 15 milligr.), while rabbits and cats were more sensitive to the toxic action. In guinea-pigs and rabbits infected with glanders bacilli very small doses had a favorable influence upon the prog- ress of the infection, and in healthy guinea-pigs a cer- tain degree of immunity was induced by the repeated in- jection of small doses. In a subsequent paper (1894) Bonome reports that he has had favorable results in the treatment of chronic glanders in man by doses of y^ to ^V c.c The first dose is said to have caused an elevation of temperature, head- ache, polyuria, etc., but upon repeating the dose after two or three daj^s a decided improvement of the general symptoms followed. Chenot and Picq (1892) claim to have cured glanders in guinea-pigs by injections of blood-serum from the ox, which animal has an immunity from the disease. They also state that the blood-serum of the ox is germicidal for the glanders bacillus. Guinea-pigs treated with ox- serum, either before or after infection, recovered in seven cases out of ten. ^lien inoculated with Aery virulent cultures, Avhich usually killed these animals in five days, the animals are said to have survived from twentj'-one to forty-two days. Bonome (1894) reports his success in curing infected guinea-pigs by means of filtered cultures made in the GLANDERS. 181 blood-serum of the ox. He was not, however, successful in accomplishiug- this result with mallein made in the usual way. BIBLIOGRAPHY. Babes, A. : Note sur une substance isolee ties cultures du bacille de la morve. Arcb. de Med. exper. et d'Aiiat. patholog., 1892, No. 4. Babes, A., et Motoc, A. M. : Sur les substances chimiques produites par le bacille de la morve. Ann. de I'lnstitut de Patliol. et de Bacteriol. de Bucbarest, ii., 63, 1893. Bononie, A. : Neue Beobacbtungen iiber die diagnostiscbe und tbera- peutiscbe Wirkung der Stollwecbselprodukte des Rotzbacillus bei der Rotzinfektion des Menscben und der Tiere. Dtscbe. med. Wcbscbr., 1894, Nos. 86-38, p. 703, 725, 744. Chenot et Picq. : De Taction bactericide du serum de sang de bovides sur le virus morveux, etc. Compte- rendu, Soc. de Biol., 1892, 26 Mars. Finger : Zur Frage der Immunitat und Pbagocytose beim Rotz. Zieg- ler's Beitrage zur patbol. Anat., Bd. VI., 1889, Heft 4. Fotli : Ueber die praktiscbe Bedeutung des trockenen Malleins. Deut- scbe Zeitscbr. fur Tiermed., XX., 223, 1894. Die " Versucbe mit der Anwendtmg des Malleins in der russis- chen Armee." Centralbl. f. Bakteriol, XVI., 508, 1894. Ueber Mallein. Zeitscbr. f. Veterinarkunde, IV., 435, 1892. Hutyra, F., u. Preisz, H. : Ueber die diagnostisclien Wert des Malleins. Dtscbe. Ztscbr. f. Tiermed., 1894, Bd. XX., p. 309. Kresling, K. : Sur la preparation et la composition de la malleine. Arch. d. Sci. Biolog., publ. par I'lnst. Imp. de Med. Exper. a St. Petersbourg, 1892, p. 711. Preusse : Versucbe mit Rotzl^nnpbe — Mallein. Berl. tbierartzl. Wcb- scbr., 1891, No. 29. Suchanka, F. J. : Impfversuclie rait Mallein. Mtsb. f. prakt. Tierbeilk., 1894, p. 481. Scliindelka, H. : Einige Erfabrungen tiber die Anwendung des Mal- leins als diagnostiscbes Mittel. Oester. Ztschr. f. wissen. Veterin- arkunde, V.^97, 1894. De Sebweinitz and Kilborne : The Use of Mallein for the Diagnosis of Glanders in Horses, etc. Journal of Comparative Med. and Vet- erinary Archives, 1892, p. 643. Schiitz : Malleinversuche. Arch. f. -wissensch. u. prakt. Tierbeilk., 1894, No. 6, p. 448. Straus : Sur la vaccination centre la morve. Compte-rendu, Acad, des Sci., t. cviii., p. 530. YII. HOG CHOLERA. Reseaeches made by the bacteriolog-ists connected with the Agricultural Department (Bureau of Animal In- dustry) have established the fact that two distinct dis- eases of swine, due to different bacilli (one motile and the other not) prevail in epidemic form in various parts of the United States. The diff'erentiatiou of the bacillus of hog- cholera was made in 1885, and a report by Theo- bald Smith, relating- to this infectious disease of swine, has recently been ijublished (1894). The bacillus pro- duces in swine a disease in which the principal pathologi- cal lesions are found in the large intestine, the mucous membrane of which is more or less ulcerated and ne- crosed ; in some cases there is at the same time a pneu- monia of limited extent. The bacillus kills rabbits, mice, and guinea-pigs when injected subcutaneously or introduced into the stomach. A small dose is fatal to a rabbit in about seven days, but larger doses kill in a shorter time, and it is more promptly fatal when injected into the circulation. Sev- eral varieties of this bacillus have been encountered l)y the bacteriologists of the Agricultural Department ; these differ chiefly in pathogenic virulence, and the curious fact is stated that the variety first studied, after artificial cultivation for six and a half years, has not lost its patho- genic power. It still kills rabbits when injected into the HOG CHOLERA. 183 circulation in small amounts (0.12 c.c). All of tlie varie- ties closely resemble tlie well-known Bacillus coli commu- nis, which has its normal habitat in the intestine of healthy men and animals. According- to Smith the ba- cillus {B. typhi murium) discovered by Loffler in 1890, which causes a fatal infectious disease in mice, is nearly allied to the hog-cholera bacillus, and should be included in the same group — which may be denominated the " colon group." The experiments thus far made with reference to pro- tective inoculations against hog- cholera have not given very satisfactory results. Selander and Metchnikoff have reported success in immunizing rabbits, but according to Smith their experiments were made with the bacillus of swine plague, and not with that of hog cholera as they supposed. The following conclusions have been formu- lated by Smith as a result of his extended experiments : " 1. It is possible to produce immunity toward hog- cholera and swine-plague bacteria in the very susceptible rabbit and the less susceptible guinea-pig. In the rabbit the only promising method of immunization toward hog- cholera is the use of gradually augmented doses of atten- uated cultures. "2. Immunization toward swine -plague is produced artificially with much greater ease than toward hog- cholera bacteria. " 3. The blood-serum of animals protected against hog cholera and swine plague is almost as efficacious in pro- ducing immunity soon after treatment as the bacterial products obtained from cultures. " 4. Different degrees of immunity in both hog cholera and swine plague lead to different forms of the inocula- tion disease. The greater the immunity short of com- plete protection the more prolonged and chronic the disease induced subsequently by inoculation. 184 IMMUNITY AND SERUM-TIIERAPY. " 5. Pathogenic bacteria may remain in the org-ans of inoculated animals some time after apparently full recov- ery. Their presence may or may not be associated with lesions recognizable by the naked eye. " 6. The toxicity of sterilized cultures appears to be di- rectly proportional to the number of bacteria in the in- jected fluid." The experiments of Moore, reported in Bulletin No. 6 of the Bureau of Animal Industry, show that the bacillus of hog cholera does not become attenuated by being passed through rabbits, and that in the experiments of Metchni- koff, whicli led him to conclude that this is the case, the bacillus of swine-plague, and not that of hog cholera, was used. De Schweinitz has studied the chemical products of the hog-cholera bacillus (1890) and has obtained from the cultures cadaverin, methylamin, a ptomaine (" sucholo- toxin"), and an albumose (" sucholoalbumiu "). Novy (1890) has also obtained, by Brieger's method, a basic toxic substance (" susotoxin ") which kills rats in the dose of 0.125 to 0.25 c.c. He also obtained from con- centrated cultures, by precipitation with absolute alcohol, a toxalbumin which, when dried, killed rats in three or four hours in the dose of 0.05 to 0.01 gm. Both of the authors nam^ed obtained experimental evi- dence indicating that these toxic substances obtained from cultures, when given to susceptible animals in non- lethal doses, cause them to be immune from the patho- genic action of small quantities of a culture of virulent hog-cholera bacilli. BIBLIOGRAPHY. Novy : The Toxic Products of the Bacillus of Hog Cholera. Philadel- phia Medical News, 1890, p. 231. HOG CHOLERA. 185 Salmon : Results of Experiments with Inoculation for the Prevention of Hog Cholera. Farmers' Bulletin, No. 8, Agricultural Department, Washington, 1892. De Schweinitz : A Preliminary Study of the Ptomaines from the Cult- ure Liquids of the Hog-cholera Germ. Philadelphia Medical News, 1890, p. 237. Smith : Zur Kenntniss des Hogcholerabacillus. Centralbl. fiir Bakte- riol., Bd. IX., 1891, pp. 253, 307, 339. The Hog-cholera Group of Bacteria. Bulletin No. 6, 1894, Bu- reau of Animal Industry, Washington. HOG ERYSIPELAS. The disease known in Germany as Rothlauf, and in France as rouget, is due to a slender bacillus, which is, ap- parently, identical with Koch's bacillus of mouse septicae- mia, which he first obtained by inoculating- mice with putrefying blood or flesh infusion. The bacillus of hog- erysipelas {Rothlauf) was first accurately described by Loffler and Scliutz (1885), althoug-h Pasteur had previously ascribed the etiology of this disease to a minute bacillus — no doubt the same — and had proved the practicability of j)rotecting- swine from the ravages of the disease by inoculations with an attenuated virus. This bacillus is l^athogenic for swine, rabbits, white-mice, house-mice, pigeons, and sparrows. Guinea-pig-s, field-mice, and chickens are immune. Swine become infected hy the ingestion of food containing- the bacillus, and usually die within two or three days. Pasteur's first studies relating to the etiology of " i^ougef'' were made, in collaboration with Chamber- land, Eoux, and Thuillier, in 1882. Pasteur found that the virulence of his cultures was increased by passing them through j)ig-eons and diminished by passing them through rabbits. By a series of inoculations in rabbits he obtained an attenuated virus suitable for protective inoculations in swine. In practice he recommended the use of a mild virns first, and after an interval of HOG ERYSIPELAS. 187 twelve days of a stronger virus. These inoculations liave been extensively practised in France, and the fact that immunity may be established in this way is well demonstrated. There has been some doubt, how- ever, as to the practical value of the method, as its appli- cation has been attended with some loss, and there ap- pears to be danger that the disease may be spread by the alvine discharges of inoculated animals. In a region where the annual losses from the disease are consider- able, and where the soil is, perhaps, thoroughly infected with the bacilli, i^rotective inoculations probably afford the best security against loss. But when it is practica- ble to stamp out the disease by quarantine of infected animals, disinfection of localities in which cases have occurred, and strict attention to cleanliness, this will probably be found the best method of combating the malady. In a recent paper (1894) Chamberland states that in the last seven years, during which time protective inocula- tions have been practised in France on a large scale, the mortality from rouget has been reduced to 1.45 per cent., whereas before these inoculations were practised the mortality from this disease was about twenty per cent. Losses amounting in some instances to as much as ten per cent, have resulted from the inoculations. These are ascribed by Chamberland to secondary infection, through the inoculation wound, with other pathogenic bacteria. Jakobi (1888) reports the results of inoculations made in 1887 and 1888 with " vaccines " obtained from Pasteur's agent in Paris. His results agree with those previously reported by Lydtin in showing a smaller loss, as a result of the inoculations, among young pigs than among older ones— over sixteen weeks. The loss among young pigs 188 IMMUNITY AND SERUM-TIIP:RAPV. was only 1.3 per cent. The animals which survived sub- sequently escaped infection, while others not inoculated, associated with them, succumbed to the disease. Hut3a'a has given the following statistics of inocula- tions made in Hungary during- the year 1889, with "vac- cines " obtained from the Pasteur laborator}^ in Vienna : 48,637 pigs Avere inoculated on 117 different farms. Of these 143 (0.29 per cent.) died between the first and sec- ond inoculation. After the second inoculation 59 animals died (0.1 per cent.). During the year following the inoc- ulations 1,082 inoculated pigs died of RolJilovf. Before the inoculations the annual loss in the same localities is said to have been from ten to thirty per cent. Upon one farm 220 pigs which had been inoculated Avere associated with 1,500 not inoculated. The loss among the latter was fifty per cent., among the former 2.27 per cent. In a later communication (1894) Jakobi gives the fol- lowing results of inoculations made since by the same method : 1889, inoculated 133, loss, 5 ; 1890, inoculated 151, loss, 2 ; 1891, inoculated 158, loss, ; 1893, inoculated 223, loss, ; 1894, inoculated 145, loss, 4. Total inoculated, 1,036 ; total loss, 14. These inoculations were made upon 19 different farms, and principally upon pigs less than four months old. The inoculated pigs were isolated to prevent the communication of the disease to other uii- protected pigs. Inoculations with Blood-serum of Immune Animals. The experiments of Lorenz, commenced in 1891, seem to establish the fact that there is an antitoxin in the blood of animals which have an acquired immunity against this disease which may be used for producing HOG ERYSIPELAS. 189 immunity in other animals, or for the cure of the disease in animals already infected. In his latest communica- tion (1894) Lorenz says : " When I read in the journals of the discovery of Beh- rinsT and Kitasato that the blood of animals immunized ag-ainst tetanus, when injected beneath the skin of other animals, gave them an immunity ag-ainst tetanus, I had in my possession rabbits which were immunized against Rotldauf. I took from one of these some blood from the ear vein, injected it under the skin of a mouse, inoculated this later with a Rothlavf culture, and made the discovery, in this and a series of subsequent experiments, that the blood of an animal immune against Rotldauf contains an immunizing substance. I further ascertained that this substance is found only in the blood-serum, and not in the solid portions of the l)ody organs, etc., and with the exception of the blood was found only in the secretions of serous membranes. I also found that the immunizing substance is only to be found for a certain time after re- newed infection in the immune animals, and that it grad- ually disappears, without the loss of iniTiiunity in the animal, however. Finally, I discovered that the animals into which one injects blood-serum from immune animals do not acquire a lasting immunity, but are only immune for a relatively short time." In experiments made in 1893 and 1894, with a view to producing immunizing serum for protective inoculations on a large scale, Lorenz met with some disappointments ; but he proposes to renew his attempts and hopes to avoid the difficulties which have been brought to light by ex- perience, one of which he states as follows : " When an animal already immunized against Rothlavf receives an injection of a considerable quantity of ds cult- ure of the bacillus, in order to cause the production in 190 IMMUNITY AXD SERUM-THERAPY. its blood of a serum of liig-h therapeutic value, the ani- mal bears these iujections without anj" notable reaction. But its blood-serum contains during- the following dajs, besides the immunizing substance, also poisonous sub- stances, and blood which is taken too soon (twentj'-four hours) after the injection has a toxic action upon animals which are already infected. If this poisonous serum is injected into a mouse which has been infected two days before with Rothlaufh&itiiW, in the dose of about 0.05 c.c, death occurs in a few hours, even when scarcely any evidence of sickness had been observed before the injec- tion." The fact that mice infected with this bacillus may be cured by injecting into them blood-serum from an im- munized rabbit has also been demonstrated by F. Klem- perer (1892). In his experiments with the bacillus of mouse septicaemia, and with Friedlander's bacillus, he found that serum from an immune rabbit may be used to immunize mice and also to cure them after infection, while serum from a non-immune rabbit has no such ac- tion. The immunity produced in this way was found to be specific. That is, animals immunized against the pathogenic action of one of these bacilli were not pro- tected against infection by the other. The " heilserum " when added to cultures in vitro did not prove to have any special bactericidal action. BIBLIOGRAPHY. Emmerich und Tsuboi : Versuch der Immiinisieruug von Schweinen gegen Rothlauf. Dtsche. tierarztl. Wchschr., 1893, p. 127. Hafner : Die Schutzimpfung gegen den Rothlauf der Schweine. Bad. thierarztl. Mitth., 1889, p. 17. Hess iind Guillebeau : Zur Schutzimpfung gegen Schweineseuche. Schweitzer Archiv fiir Thierheilk., Bd. XXVIII., 1886, Heft 3. HOG ERYSIPELAS. 191 Jakobi : Beitrag zur Scliutzimpfunggegeii den Rotblauf der Schweiiie. Eerl. thiei-arztl. Wchsclir., 1888, No. 50. Beitrag zur Pa.steursclien Scliutzimpfuiig gegeu Rothlauf der Schweine. Berl. thieriirztl. Wcbschr., 1894, p. 234. Kitl : Beitrage zur Kenntniss des Slabschenrothlauf der Scbvveiiie und dessen Scbutzimpfung. Revue ftir Heilk. und Tbierzucbt., 1886. Uiitersucbuiigen tiber den Stabscbenrotblauf und dessen Scbulz- inipfung. Centralbl. fur Bakteriol., Bd. II., 1887, p. 693. Lorcuz : Immunisierungversucbe gegen Scbweinerolbhuif. Bad. tbier- arztl, Mittb., 1893, p. 33. Ein Scbutzimpfungsverfabren gegen Scbweinerotblauf. Cen- tralltl. f. Bakteriol., XIII., 357, 1893. Sebutzimpfungsversucbe gegen Scbweinerotblauf mit Anwen- dung eines aus Blutserum immunisierter Tiere hergestellten Impf- praparates. Dtscbe. lierarztl. Wcbschr., 1894, p. 9. Sebutzimpfungsversucbe gegen Scbweinerotblauf. Centralbl. fiir Bakteriol., XV., 278, 1894. Lj'dtin : Scbutzimpfungen gegen den Rotblauf der Scbweine. Bad. tbierarztl. :Mitt., 1886, No. 9. Pasteur : Le rouget du pore ; avec la collaboration du MM. Cbamber- land, Roux et Tliuillier. Conipte-reudu, Acad, des Sci., xcv., 1882, p. 1120. Pasteur et Tbuillier : Bull, de I'Acad. de Med. Paris, xcvii., 1883. Schiitz : Ueber den Rotblauf der Scbweine und die Impfung derselbeu. Arbeit, aus dem K. Gesundbeitsamte, Bd. I., 1885. IX. HYDROPHOBIA. Notwithstanding the extended researches made, espe- cially in Pasteur's laboratory, the etiology of hydro- phobia still remains unsettled. It has been demon- strated by experiment that the virus of the disease is lo- cated in the brain, spinal marrow, and nerves of animals which have succumbed to the disease, as w^ell as in the salivar}^ secretions of rabid animals ; and that the disease may be transmitted by intravenous inoculation, or by in- troducing- a small quantitj^ of virus beneath the dura mater, with greater certainty than by subcutaneous in- oculations. But the exact nature of this virus has not been determined. The fact that a considerable interval elapses after inoculation before the first symptoms are developed indicates that there is a multiplication of the virus in the body of the infected animal ; and this is fur- ther shown by the fact that after death the entire brain and spinal marrow of the animal have a virulence equal to that of the material with which it was inoculated in the first instance. The writer's experiments (1887) show that this vi^'ulence is neutralized by a temperature of 60° C, maintained for ten minutes — a temperature which is fatal to all known pathogenic bacteria in the absence of spores. But recent experiments show that certain toxic products of bacterial growth are destroyed by the same temperature. We are, therefore, not justified in assum- HYDROPHOBIA. 193 ing- tliat the morbid phenomena are directly due to the j)resence of a living- microorganism ; and, indeed, it seems probable, from what we already know, that the symptoms developed and the death of the animal are due to the action of a potent chemical poison of the class known as toxalbumins. But, if this is true, we have still to account for the production of the toxic albuminoid substance, and, in the present state of knowledge, have no other way to explain its increase in the body of the infected animal than the supposition that a specific, liv- ing' germ is present in the virulent material, the intro- duction of which into the body of a susceptible animal gives rise to the morbid phenomena characterizing- an attack of rabies. Pasteur and his associates have thus far failed to de- monstrate the presence of microorganisms in the virulent tissues of animals which have succumbed to an attack of rabies. Babes has obtained micrococci in cultures from the brain and spinal cord of rabid animals, and states in his article on hydrophobia in " Les Bacferies " (second edition, p. 791) that pure cultures of the sec- ond and third generation induced rabies in susceptible animals ; but his own later researches do not appear to have established the etiological relation of this micro- coccus. Gibier (1884) has reported the presence of spherical refractive granules, resembling micrococci, in the brain of rabid animals, which he demonstrated by rubbing up a little of the cerebral substance with distilled water. As these supposed micrococci did not stain with the usual aniline colors and were not cultivated, it appears very doubtful w^hether the refractive granules seen were really microorganisms. 13 194 IMMUNITY AND SERUM-THERAPY. Fol (1885) claims to have demonstrated the presence of minute cocci, 0.2 /* in diameter, in sections of spinal cord from rabid animals, by Weigert's method of staining-. The cords were hardened in a solution of bichromate of potash and sulphate of copper, colored with a solution of hsematoxylon, and decolorized in a solution of ferrocya- nide of potash and borax. The writer (1887) has made similar prei^arations, carefully following- the method as described by Fol, but was not able to demonstrate the presence of micro- organisms in the numerous sections made. Nor have the observations of Fol been confirmed by the re- searches of other bacteriologists who have given their attention to the subject since the publication of his paper. Pasteur first announced his success in reproducing rabies in susceptible animals by inoculations of material " from the medulla oblongata, the frontal lobes of the cerebral hemispheres, and the cerebrospinal fluid " in a communication to the Academy of Sciences made on May 30, 1881. At the same time he reported his success in the discovery of " a method for considerably shorten- ing the period of incubation in rabies, and also of repro- ducing the disease with certainty." This was by inocula- tions, made after trephining, upon the surface of the brain with material obtained from the brain of a rabid animal. Dogs inoculated in this way developed rabies in the course of two weeks, and died before the end of the third week — sometimes of furious rabies and sometimes of the paralytic form of the disease. In a second communica- tion (December 11, 1882) Pasteur reports his success in communicating the disease by the intravenous injection of virus from the central nervous system ; also the ex- HYDROPHOBIA. 195 perimental demonstration of the fact that all forms of rabies may be produced by the same virus ; also that all portions of the spinal cord of rabid animals are virulent, as well as all parts of the brain ; also that an animal (dog-) which had recovered from a mild attack after in- oculation proved to be subsequently immune, and that " this observation constitutes a first step toward the discovery of the prophylaxis of rabies." On February 25, 1884, many important facts are stated which had been developed during the continuous study of the disease, and among- others the fact that by passing the virus through a series of animals of the same species a fixed degree of virulence is established, for .each susceptible species, as shown by a definite and uniform period of incubation. By this method a virus had been obtained which pro- duced rabies in rabbits in seven or eight days, and an- other which caused the development of the disease in guinea-pigs in five or six days after inoculation. In a subsequent communication (May 19, 1884) evidence is given to show that by successive inoculations in monkeys the period of incubation is prolonged, and that the at- tenuated virus obtained from a monkey, after several suc- cessive inoculations in this animal, when inoculated into the dog, no longer produces fatal rabies ; and that dogs so treated are subsequently immune. In his address before the International Medical Con- gress at Copenhagen (August 11, 1884), after a review of the facts developed during his experimental researches made during the preceding four years, Pasteur gives an account of the test made by a commission, appointed by the Minister of Public Instruction, to determine the effi- cacy of his method as applied to the protection of dogs. He says that he gave to the commission nineteen dogs 196 IMMUNITY AND SERUM-THERAPY. which had been rendered refractory against rabies by preventive inoculations. These nineteen dogs and nine- teen control animals, obtained from the pound without any selection, were tested at the same time. The test was made upon some of the animals of both series by inocu- lation with virulent material upon the surface of the brain, and upon others by allowing them to be bitten by rabid dogs, and upon still others by intravenous inoc- ulations. Not one of the protected animals developed rabies ; on the other hand, three of the control dogs out of six bitten by a mad dog developed the disease, five out of seven which received intravenous inoculations died of rabies, and five which we^-e trephined and inoculated on the surface of the brain died of the same disease. In a subsequent report the commission, of which M. Bouley was president, stated that twenty-three protected dogs which were bitten by ordinary mad dogs all remained in perfect health, while sixty-six per cent, of the control animals, bitten in the same way, developed rabies with- in two months. In his communication of October 26, 1885, Pasteur re- ports his discovery of the fact that the virulence of the spinal cord of a rabbit is gradually attenuated by hang- ing it in a dry atmosphere, and is finally entirely lost ; also that he had been able to make a practical applica- tion of this discovery in the protection of dogs by means of successive inoculations beneath the skin of an emul- sion of spinal marrow attenuated in this way. The first inoculation was to be made with a portion of spinal cord which had been kept long enough to deprive it of all virulence, and this was followed by daily inoculations with more virulent material until, finally, material was used from a cord only a day or two old. HYDROPHOBrA. 197 With reference to his first inoculations in man, Pasteur says: " Making use of tliis method, I had already made fifty dogs of various races and ages immune to rabies, and had not met Avitli a single failure, when, on the 6th of July, quite unexpectedly, three persons, residents of Alsace, presented themselves at my laboratory." These persons were Theodore Vone, who had been bit- ten on the arm on July ■Ith, Joseph Meister, aged nine, bitten on the same day by the same rabid dog, and the mother of Meister, who had not been bitten. The child had'been thrown down by the dog and bitten upon the hand, the legs, and the thighs, in all in fourteen different places. Pasteur commenced the treatment on July 6th, by injecting beneath the skin of this child an emulsion of cord which had been kept for fourteen days; this was followed by twelve more inoculations made on successive days with cord of increasing degrees of virulence — the last with cord a day old. On March 1, 1886, Pasteur re- ported to the Academy of Sciences the fact that the boy Meister remained in good health and gave detailed infor- mation with reference to a number of cases which had since been treated by the same method. With reference to the duration of the immunity result- ing from these inoculations Pasteur says (188G) that out of fourteen dogs inoculated with " ordinary street virus, ' by trephining, at the expiration of a year after the pro- tective inoculations had been practised, eleven resisted ; out of six tested in the same way at the end of two years two proved to be immune. In November, 1886, Pasteur communicated to the Acad- emy of Sciences the results of his experiments with refer- 198 IMMUNITY AND SERUM-THERAPY. ence to a modification of his metliod as at first employed — the so-called intensive method. This modification con- sisted in making the inoculations with cords of increasing virulence in more rapid succession. The method followed at Odessa, as reported by Gama- leia (1887), is shown below, the day being given above and age of the cord below. 1 2 3 4 5 6 1 8 9 10 10-9 8-7 6-5 4-3 2-10 8-6 4 2 14-13 12-11 Since the adoi^tion of this method and the use of larger quantities of virus, according to Gamalei'a, there have been no deaths among those inoculated, numbeadng more than two hundred at the time the report was made. The author last referred to concludes from his experience that " the mortality diminishes in direct relation to the quantity of the vaccine injected." Bujwid (1889) reports a total of 670 inoculations, with 9 deaths, made at Varsovio during the years 1886, 1887, and 1888. His method is shown below. 12-10 8-6' The results of inoculations made at the Pasteur Insti- tute in Paris during the years 1886 to 1890 are given in the following table : Year; Number Treated. Died. MortaUty. 1886 2,671 1,770 1,622 1,830 1,540 25 13 9 6 5 0.94 1887 0.73 1888 0.55 1889 0.83 1890 0.32 Total 9,433 58 0.61 HYDROPHOBIA. 199 In the following- table, A includes all persons treated wlio had been bitten by an animal proved to be rabid ; B, persons bitten by animals examined by veterinary snr- g-eons and pronounced rabid ; C, persons bitten by ani- mals suspected of being- rabid. The figures relate to the year 1890 : Number Treated. Died. Mortality. A 416 909 215 4 1 B 0.44 (; 46 Bordoni-Uffreduzzi g-ives the following statistics with reference to the inoculations practised at the Pasteur In- stitute in Turin during the years 1886 to 1891 : 81 persons were inoculated by the method first proposed by Pasteur, with a mortality of 2.46 per cent.; 925 persons were subse- quently inoculated by the same method, but with larg-er doses of virus, with a mortality of 1.72 per cent. Finally, 338 persons were inoculated with still larg-er doses, with a mortality of 0.29 per cent. At the Pasteur Institute in Palermo the number of per- sons inoculated in the four years prior to 1891 was 662, with a mortality among the inoculated of 0.6 per cent. In Bologna (1890) 210 persons bitten by dog's undoubtedly mad were inoculated, with a mortality of 0.47 per cent. In the Pasteur Institute at Naples 810 persons were treated during the years 1886 to 1892, with a mortality of 0.86 per cent. During the year 1891, 1,564 persons were inoculated at the Pasteur Institute in Paris, with a total mortality of 0.57 per cent. In 324 of these cases the animal which in- flicted the bite was proved to be rabid by experimental inoculations. 200 IM3IUNITY AND SERUM-THERAPY. Horsely (1889) lias made a comparison of the results obtained by the " intensive treatment " as compared with those by the treatment first employed, and says : "It is evident that the intensive treatment is very suc- cessful in coping with the worst cases, and that, instead of being- itself a source of death, as asserted by those who gain notoriety and subsistence by vilifying and misrep- resenting scientific progress, it is a powerful agent in saving life." The following table is given by Horsely " as showing the contrast between the old or simple treatment and the intensive treatment : " Simple Treatment, 1886. Intensive Treatment, 1888. Odessa 3.39 per cent. 0.64 per cent. Warsaw 4.1 " " 0.0* " " Moscow 8.2f " " 1.6 " " Perdrix (1890), in an analysis of the results obtained at the Pasteur Institute in Paris, calls attention to the fact that the mortality among- those treated has diminished each year and ascribes this to improvement in the meth- od. He says : " At the outset it was difiicult to know what formula to adopt for the treatment of each particular case. Uj)on consulting the accounts of the bites in persons who have died of hydrophobia notwithstanding the inoculations, we have arrived at a more precise determination as to the treatment suitable for each case, according to the gravity of the lesions. In the cases with serious wounds we in- ject larger quantities of the emulsion of cord and repeat * The figures include sixteen months' work, and thirtj- individuals bitten in the face — four by wolves. + This unusually high rate was found to be due to imperfections in the man- ner of preparing the cords for the inoculation material. HYDROPHOBIA. 201 the inoculations with the most virulent material. For the bites upon the head, which are especially dangerous, however slight their apparent gravity may be, the treat- ment is more rapid, and, above all, more intensive — that is to say, the virulent cord is injected several times." The statistics arranged with reference to the location of the bite are given by Perdrix as follows : Bitten upon the head, 684 ; died, 12, = 1.75 per cent. " hands, 4,396; " 9, = 0.2 " " " limbs, 2,839; " 5, = 0.17 " " Other methods of making susceptible animals immune against hydrophobia have been proposed and proved by experiment to be successful. Thus Galtier in 1880-1881 claimed that the sheep and the goat could be protected by intravenous injections of the virus of rabies, and more recent experiments fully confirm this. Protopopoff (1888) by injecting an emulsion of cord from a rabid animal into the circulation of dogs succeeded in protecting them from hydrophobia as a result of a subsequent inoculation with virulent material upon the surface of the brain. He in- jected into a vein, at intervals of three daj's, 1 c.c. of an emulsion of cord — first of six days, second of three days, third of one da3^ Roux had previously accomplished the same result by a single intravenous injection of a larger quantity (35 c.c.) of cord which had been kept for five or six days. In discussing his results Roux calls attention to the fact, which had been developed during his experiments, that the virulence of the spinal cord of rabid animals does not depend entirely upon the length of time it has been kept, but that large doses of cord kept as long as twelve days will sometimes produce hydrophobia when injected into the circulation of dogs, when smaller doses of cord kept five or six days prove to be inofi'eusive. 202 IMMUNITY AND SERUM-THERAPY. He supposes that during- desiccation the virus may not be equally acted upon throughout the cord, but that cer- tain " islands " in the centr. J portion may remain living- and ^druleut when all the rest has been modified. A IDractical point with reference to the preservation of vir- ulent material is referred to by Roux in a note published in the " Annales of the Pasteur Institute." This is the fact that when preserved in glycerine jDortions of the cen- tral nervous system retain their virulence for considera- ble time. Other forms of virus, e.g., vaccine, may also be preserved in the same way. Centanni (1892) has succeeded in making rabbits im- mune by inoculating them with an attenuated virus ob- tained by subjecting virulent material to the action of an artificial gastric juice. After digestion for less than twelve hours the virus still kills rabbits, when inoculated beneath the dura mater, but the period of incubation is considerably prolonged. After from twelve to twenty hours' digestion it no longer kills ral)bits, but causes an infection, from which they recover, and after which they are immune. Sent n t-the > ajyy. Tizzoni and Centanni (1892) have reported success in the treatment of infected rabbits by the use of blood- serum from immune animals of the same sjiecies — immu- nized by the " Italian method " above described. The animals experimented upon were inoculated with a " street virus " which produced paralytic rabies in rab- bits and caused their death in from fourteen to eighteen days. The blood-serum was obtained from rabbits which had been proved to be immune by resisting inoculations HYDKOPHOBIA. 203 of virus of full strength on tlie surface of the brain. The blood-serum, in doses of 3 to 5 c.c, was injected subcu- taneously, or into the peritoneal cavity, or into the circu- lation. Injections were made into each animal (in all from 11 to 26 c.c.) after the first symptoms of paralytic rabies had appeared (on the 7th, 10th, lltli, and 14th day after infection). Four rabbits treated in this way fully recovered. In a subsequent experiment the bacteriolo- g-ists named treated three rabbits with a dry antitoxin obtained by precipitation from the blood-serum of im- mime rabbits. The precipitate was obtained by adding- 1 part of serum to 10 parts of alcohol, and was dried 171 vacuo. This dried precipitate, in doses of 0.18 to 0.25 gm., was dissolved in sterilized water and injected as in the previous experiment. Commencing- on the eighth day after infection five or six doses were g-iven — in all 0.9 to 1.3 g-m. All of the animals treated recovered, wdiile all of the control animals died. Babes had previously (1889) reported successful results in conferring- immunity upon susceptible animals by injections of blood-serum from immune animals. Tizzoni and Schwartz, in pursuing this line of investi- gation (1892), report that while the blood-serum of im- mune rabbits neutralizes the " fixed virus " of rabies m vifi'o, after short contact (five hours), the blood-serum of immune dogs has but slight antitoxic potency. The im- munizing substance in the rabbit serum does not dialyze, is soluble in g-lycerin, is precipitated by alcohol, and in general behaves like a globulin. In subsequent experi- ments Tizzoni and Schwai-tz used blood-serum from dogs and rabbits immunized by Pasteur's method. The blood was drawn from the carotid of the immune animals, and the serum from the same, mixed with virulent spinal mar- 204 IMMTTNITY AT^D SERUM-TIIEKAPY. row in the form of a liomog-eneous emulsion, obtained by crushing' and pressing- through linen. These experiments corresponded with those iireviously made as to the supe- rior antitoxic power of rabbit-serum, which, after five hours' contact, neutralized the virulence of the emulsion of cord. By the injection of serum from an immune rabbit, in doses of 5 c.c, into the circulation of other rabbits, they were, as a rule, made immune. The immunizing- sub- stance (antitoxin) was shown by other experiments to be present only in the blood. Extracts from the liver, spleen, kidneys, or muscles gave a negative result. In their latest communication (1894) Tizzoni and Cen- tanni give an account of further exj)eriments made prin- cipally upon sheep and dogs. By repeated inoculations they succeeded in obtaining from these animals a serum having an immunizing value of 1 to 25,000 or more, and from this a precipitate was obtained estimated to have a value of 1 to 300,000, and which in doses of 0.23 gm. (of the dried precipitate), dissolved in five times its weight of water, ought to be a sufficient dose to protect a man from the development of hydrophobia after being bitten by a rabid animal. The authors named believe that inoculations with this antitoxin would be reliable for man, and that they would possess decided advantages over Pasteur's method of in- oculation. These advantages are specified as follows : " Applicability at any time during the period of incu- bation up to the moment of the appearance of symptoms of rabies ; absolute absence of virulence and of any in- jurious action ; very rapid treatment by the injection of one or several small doses of material ; complete solubil- ity and consequently jirompt absorption of the material injected and its easy preservation in a dry condition." HYDROPHOBIA. 205 Finally, the authors say that they are engaged in pre- paring the antitoxin on a sufficient scale to enable the test to be made upon man, and that for this purpose they are using sheep as the most suitable animals for the pur- pose. BIBLIOGRAPHY. Babes et Lepp : Reclierches siir la vaccination antirabique. Ann. de I'lust. Pasteur, vol. iii., 1889, p. 384. Babes, V. : Ueber die ersten erfolgreiclien Iinpfungen gegen Huuds- wutli mittels Blut immunisirter Thiere. Dtsche. med. Wchsclir., 1893, No. 41. Bardacli : Sur la vaccination intensive des chiens inocules de la rage par trepanation. Ann. de I'lnst. Pasteur, vol. i., 1887, p. 84. Bujwid : La methode Pasteur a Varsovie. Ann. de I'lnst. Pasteur, 1889, p. 177. Centanni, E. : II Metodo italiano di vaccinazione antirabbica. Ri- forma Med., 1893, Nos. 103, 103, 104. Die spezifiscbe Immunisation der Elemente der Gevvebe. Ein Beitrag zur Keuntniss der Immunitat und der Serumtherapie bei Rabies. Dtscbe. med. Wchscbr., 1893, pp. 1061, 1115. Gamaleia : Vaccination antirabique des animaux. Ann. de I'lnst. Pas- teur, vol. i., 1887, pp. 137, 296. Galtier : Nouvelles experiences tendant a demontrer I'efflcacite des in- jections intra-veineuses de virus rabique. Compte-rendu, Acad, des Sci., evil., 1888, p. 798. Nouvelles experiences sur I'inoculation antirabique, etc. Compte- rendu, Acad, des Sci., 1888, and cvi., p. 1189. Hogyes, A. : Vaccinations centre la rage avant et apres infection. Ann. de I'lnst. Pasteur, t. iii., 1889, p. 439. Die Statistik des dritten Jahres am Budapester Pasteur-Institut. Ungar Arcb. f. Med., 1894, p. 1. Horsley, Victor : On Rabies, its Tieatment by M. Pasteur, and on tbc Means of Detecting it in Suspected Cases. British Medical Jour, nal, Feb. 16, 1889, p. 343. Nocard et Roux : Experiences sur la vaccination des ruminants coU' tre la rage, par injections intra-veineuses de virus rabique. Ann. de I'lnst. Pasteur, vol. ii., 1888, p. 341. Pasteur : Sur la methode de prophylaxie de la rage apres morsure. Compte-rendu, Acad, des Sci., t. cviii., 1889, p. 1338. Pasteur, Chamberland et Roux : Methode pour prevenir la rage aprfis morsure. Compte-rendu, Acad, des Sci., 1885 (October 36th). 206 IMMUNITY AND SERUM-THERAPY. Peter : Les vaccinations antirabiques. Journal de Micrographie, 1887, p. 449. Perdrix : Les vaccinations antirabiques a I'Institut Pasteur. Ann. de rinst. Pasteur, vol. iv., 1890, p. 129. Protopopoff : Zur Immunitat fur Tolhvutligift bei Huuden. Centralbl. fiir Bakteriol., Bd. IV., 1»88, p. 85. Ueber die vaccination der Hundc gegeu Tollwutb. Centralbl. fur Bakteriol., Bd. IV., 1888, p. 787. Ueber die Hauplursacbe der Abschwacbung des ToUwutbgiftes. Centralbl. fiir Bakteriol., Bd. VI., 1889, p. 129. Roux : Note sur un moyen de couserver les moelles rabiques avec leur virulence. Ann. de I'lnst. Pasteur, vol. i., 1887, p. 87. Note de laboratoire sur I'iinmunite conferee aux cbiens contre la rage par I'iujectiou intra-veineuse. Ann. de I'Inst. Pasteur, vol. ii., p. 479. Suzor : Hydropbobia ; An Account of M. Pasteur's System, containing a Translation of all bis Comm.uuicatious on tbe Subject, tbe Tecb- niqiie of bis Metbod, and tbe latest Statistical Results. London, 1887, pp. 231. Tizzoni, G., e Centanui, E. : Ulteriori ricbercbe suUa cura della rab- bia sviluppata. Riforma Med., 1892, No. 182. Siero antirabbico ad alto potere immunizzante, applicabile all' uomo. Riforma Med., 1893, p. 855. Serum gegeu Rabies, von bober immunisierender Kraft, auf den Menscben anwendbar. Berl. klin. Wocbenscbr., 1894, p. 189. Tizzoni et Scbwartz : La prophylaxie et la guerison de la rage par le sang des animaux vaccines contre cette maladie. Ann. de Micro- grapbie, t. iv., 1892, p. 169. La Profilassi e la cura dolla rabbia col sangue degli animali vac- cinati contro quella malattia. Riforma Med., 1892, Nos. 18 and 19. Ullmann : Ein Beitrag zur Frage tiber den Wertb der Pasteur'scben Scbutzinipfungen am Menscben. Wiener Med. Blatter, 1887, p. 1260. Vulpian : Nouvelle statistique des per.sonnes qui ont ete traitees a. I'in- stitut Pasteur, etc. Compte-rendu, Acad, des Sci., 1887 (January 28tb). Wilke : Resultate der Pasteur'scben Scbutzimpfung in russischen Insii- tuten. Hygien. Rundschau, 1894, No. 20, p. 920. X. INFLUENZA. The bacillus discovered by Pfeiffer, in 1892, is now well established as the specific cause of this disease. Bruschettini has recently (1893) reported the details of his experiments upon rabbits, for which animals this bacil- lus is pathog-enic. As a result of these experiments he has reached the following- conclusions : " 1. Eabbits may be vaccinated ag-ainst the pathogenic action of cultures of the influenza bacillus without g-reat difliculty. " 2. The best material for producing a liig-h grade oi" immunity is blood-cultures which have been filtered through the Berkenfeld filter. " 3. The blood-serum of immunized animals has strong antitoxic properties, but has no germicidal power. " 4. The serum of vaccinated animals has the power of conferring immunity upon other animals, in compara- tively small amounts — in the proportion of 1 to 42,000 of body-weight, and perhaps still less. " 5. This serum has also a decided"' curative action, and rescues rabbits from death even as late as forty-eight hours after infection by injection of a culture of the bacil- lus into the trachea." These results lead the author to hope that serum-ther- apy'may afford a method of curing this disease in man. For this purpose the blood of an immune rabbit would 208 IMMUNITY AND SERUM-THERAPY. appear to be the most promising source from which to procure an antitoxic serum. BIBLIOGRAPHY. Bruscliettini : Nuovo contribulo alio studio del bacillo dell' iuflueuza e specialmente della sua azione patogena vel couiglio. La Riformu Med., 1893, p. 8L XI. INFLUENZA IN HORSES. ScHUTZ (1887) has described a minute oval bacillus, usually associated in pairs, which appears to be the spe- cific infectious agent in the disease known in Germany as Brustseuclie. This bacillus is pathogenic for mice, rab- bits, pigeons, and guinea-pigs, but not for swine or chickens. By injection of cultures into the parenchyma of the lungs Schiitz reproduced the disease — confirmed in 1888 by Hell. Horses which have suffered an attack of infectious in- tluenza are subsequently immune, and the experiments of Hell have shown that an immunity also follows the dis- ease which results from inoculations with pure cultures of the Schiitz bacillus. The extended experiments made by the War Depart- ment of the German Government show that the disease is not produced by intravenous injections or by the in- gestion of the bacillus with the food. Infection occurs, however, when cultures are injected into the respiratory passages. Subcutaneous injections cause a painful local tumefaction, often followed by an abscess, but without the general symptoms of influenza. Experiments have been made in Germany, by Hell, Siedamgrotzki, and others, which indicate that the sub- cutaneous injection of blood-serum from immune horses may confer immunity on other horses. Hell usually in- 14 210 IMMUNITY AND SERUM-TIIERAPY. jected 40 c.c. at a time, and repeated this at intervals un- til 200 to 240 c.c. had been injected in the course of two or three weeks. He also reports the results of treatment by injections of blood-serum into the trachea in horses already infected, and thinks these injections had a favor- able influence on the course of the disease. Experiments made subsequently by Toepper have given a similar re- sult, but others have not been so fortunate, and the im- munizing- value of blood-serum injections, as practised by the authors referred to, seems to be still a matter of some doubt. Toepijer (1893) gives full directions for collecting the serum and a detailed account of results of experimen- tal inoculations made by himself and others. He prefers to inject the serum into the breast over the ensiform car- tilage. No reaction occurs after the injection. BIBLIOGRAPHY: Bnischettini, A. : Die experimentelle Immuuitat gegen Influenza. Dtscbe. med. Wcbschr., 1893, p. 790. Hell : Immunisirungsversuche mit Blutserum gegen Brustseiiche. Zeitschr. f. Veterinark., IV., 453, 527, 1892. Schiitz : Weitere Mittheilungen iiber Impfungen mit Blutserum zum Scliutze gegen die Brustseuche. Zeitschr. ffir Veterinarkunde, Bd. v.. Heft 5. Toepper, P.: Die Brustseuche der Pferde und die Bekampf ung dersel- ben durcb Impfung. Berl. tierarztl. Wcbschr., 1893, pp. 342, 853. xn. PLEURO-PNEUMONIA OF CATTLE. In a recent communication (1894) to the Central Society of Veterinary Medicine (of France), Arloing- claims that he has demonstrated the etiological relation of a bacillus lirst described by him in 1889 {Pnemjiohadllus liquefaciens hovis) to the infectious disease of cattle known as pleuro- pneumonia. The demonstration was not complete until recently, because of failure to reproduce the disease by inoculation with a pure culture of the bacillus. Arloing- asserts that he has now succeeded in accomx)lishing- this, and ascribes his earlier failures to the fact that the bacilli in his cultures were too much attenuated to produce the disease by inoculation. He has found that the bacilli as found in the serum from the lung-s become more virulent when they develop in the subcutaneous tissues of an ani- mal ; and cultures made from the bacillus procured from the subcutaneous tissues at the point where protective in- oculations are usually practised — the tail — proved to be sufficiently active to reproduce the disease when they were injected into the lung-s. Chauveau, who was pres- ent at the meeting- when Arloing read his most recent paper on the subject (June 28, 1894), states that he has seen the inoculated animals and has no doubt that the bacillus in question is the infectious agent. Although this demonstration is of such recent date, protective inoculations against this disease have long 212 IMMUNITY AND SERUM-THERAPY. been successfully practised. For this purpose serum obtained from the lungs of an animal recently dead has been employed, this having- been proved by exi^eriment to be infectious material altlioug-h the exact nature of the infectious agent present in it was not determined. Willems, who was one of the first to advocate the use of protective inoculations in this disease (1852), has re- cently given a lecture (1894) in which he has reviewed the evidence in favor of these inociilations in the disease under consideration. Various methods have been employed. Thus Willems states that the natives of the banks of the Zambeze cause animals to swallow a certain quantity of the liquid from the pleural cavity of an animal recently dead, and thus g-ive them immunity. The virus has been injected into the circulation by some experimenters, and others have proposed to attenuate it by heat. But the method which has been most extensively employed is that discovered by the Dutch settlers at the Cape of Good Hope (the Boers), and consists in inoculating- ani- mals in the tail with serum from the lung-s of an animal recently dead ; or with a virus obtained from the tume- faction produced by such an inoculation in the tail. This secondary virus was very extensively used by Lenglen, a veterinarian at Arras, who communicated his results to the Academy of Science at Paris, in April, 1863, and Wil- lems says, in his last published communication, that this is the method which he prefers. It is also the meth- od most extensively employed in Australia, into which country infectious pleuro-pneumonia was introduced in 1858. It quickly spread and has caused enormous losses. The killing of all animals, sick or suspected of being infected, was tried for several years ; but this proved to be ineffectual for stamjiing- out the disease, and the sacrifice PLEURO-PNEUMONIA OF CATTLE. 213 was SO great that this measure of prophylaxis was aban- doned. According- to Loir, attention, in Australia, was called to Willem's method of protective inoculations, in 1861, by a letter from Cape Colony published in the journals of Syd- ney and in Melbourne. " The method was at once applied both in Victoria and in New South Wales, and since that date many thousands of cattle have been inoculated. In order to obtain a sufficient supply of virus the method recommended by Pasteur in 1882 has been followed. This is described by Pasteur himself in the following words : " With a single lung we may procure sufficient virus to serve for numerous series of animals. And without hav- ing recourse to other lungs this provision may be main- tained in the following manner : It is sufficient before the supply of virus is exhausted to inoculate a young calf in the dewlap or in the shoulder. The animal dies very promptly, and all its tissues near the point of inoculation are infiltrated with serum, which is virulent, and may be collected and preserved in a state of purity." Loir prefers to obtain the virus in this way from a calf six to twelve months old, during the second week after inoculation, when the temperature of the animal has gone up to 40° to 42° C, as the virus is then said to possess the maximum degree of intensity. This vaccine seems to be- come attenuated in passing through a series of animals by inoculation, so that when it has been passed through a series of five animals it no longer produces death even when inoculated in the most dangerous localities. Loir testifies to the protective value of inoculations with this virus made in the tail of the animal, and gives the follow- ing example : A few months prior to the publication of his paper (1893), about two thousand cows were inocu- 214 IMMUNITY AND SERUM-THERAPr. lated with a virus wliicli had been passed through a series of five calves. At the moment of being- driven away they were joined by nineteen other cows not vaccinated. After being on the road for a distance of two thousand kihj- metres the animals arrived at their destination. The two thousand vaccinated were in good condition, while eight of the non-vaccinated had died of pleuro-pneumonia. In the BiiUetln of Ihe Central Sccieti/ of Veterinary Medicine of May 24, 1894, M. Robcis reports the results of inoculations made with cultures of Arloing's Pneumoha- cillus liquefaciens bovis, and with injections of pulmonary serum. His statistics with reference to the last-mentioned " legal " inoculations he has obtained from official docu- ments relating to the Department of the Seine. The total number of infected localities in this depart- ment during the years 1885 to 1891 was 1,253 ; total num- ber of contaminated animals, 18,356 ; total number inocu- lated, 18,359 ; total number of deaths prior to inoculation, 1,753 ; total number of deaths after inoculation, 2,741 ; total number of deaths due to the inoculation, 94 ; total percentage of mortality, 22.8 per cent. After discussing these and other statistics Robcis arrives at the conclusion that Arloing's method of preventive inoculations with cultures of the PnetitnohaciUns Uquefaviens bo vis gives better results than the legal method with serum from an infected animal, the total loss among animals exposed to contagion not being over twelve to fourteen per cent. Nocard (1892) says that serum from the lungs of an animal dead from pleuro-pneumonia preserves its viru- lence and usefulness as a vaccine, when mixed with half a volume of pure neutral glyceiin and half a volume of a five per cent, solution of carbolic acid. At the end of two and a half months this mixture preserved its full virulence. PLEURO-PNEUMONIA OF CATTLE. 215 BIBLIOGRAPHY. Arloing, S. : Sur les proprietes putliogenes des mati^res solubles fa- briquees par le microbe de la peripneumonie contagieuse des bovides, etc. Coinpte-reudu, Acad. desSci., cxvi., p. 166, 1893. Production experimentale de la peripneumonie contagieuse du boeuf a I'aide de cultures. Recueil de Med. veterin., 1894, p. 505. Nocard : Moyen simple de conservation du virus peripiieumonique. Bull, de la Soc. Centrale de Med. veter., xlvi., 303, 1893. Rebels, M. : Avantage de I'inoculalion preventive, facultative, centre la peripneumonie contagieuse du boeuf. Bull, de la Soc. Cent, de Med. veter., 1894 (May 34th). Semmer, E. : Ueber des Rinderpestcontagium und iiber Immuuisierung und Schutzimpfung gegen Rinderpest. Berl. tierarztl. Wchschr., 1893, p. 590. Willeras : La vaccination bovine. Bull, de I'Acad. R. de Med. de Bel- gique, 1893, p. 382. xin. PNEUMONIA. The micrococcus of cronpus pneuftionia was discov- ered by the present writer in the blood of rabbits inocu- lated subcutaneously with his own saliva in September, 1880. In 1885 this micrococcus, which I had repeatedly obtained in pure cultures from the blood of rabbits in- oculated, as in the first instance, with my own saliva, was identified with the micrococcus of the same form present in the rusty sputum of patients with pneumonia. In a paper read before the Patholog-ical Society of Philadel- phia, in April, 1885, and published in the American Jour- nal of the Medical Sciences on July 1st of the same year, I say : " It seems probable that this micrococcus is concerned in the etiology of croupous pneumonia, and that the in- fectious nature of the disease is due to its presence in the fibrinous exudate into the pulmonary alveoli." This has since been fully established by the researches of Frankel, Weichselbaum, Netter, Gameleia, and many others. Frankel first discovered this micrococcus in his own salivary secretions in 1883, and his first paper relat- ing to its presence in the exudate of croupous pneu- monia was published on July 13, 1885, i.e., thirteen days after the publication of the paper from which the above quotation is made. Under these circumstances the PNEUMONIA. 217 writer feels justified in ag-ain calling- attention to his priority in the discovery of this important pathog-enic micrococcus, and in objecting- to its being- described as " Frankel's pneumococcus," the " diplococcus of Fran- kel," etc. In my iJaper above referred to (July, 1885) I described this micrococcus under the name of Micrococcus Pas- teuri, but in my " Manual of Bacteriology " (1892) it is described under the name of Micrococcus pneumonice croiiposce. This micrococcus is very pathogenic for mice and for rabbits, less so for guinea-pigs and for dogs. Like other pathogenic microorganisms of the same class it varies greatly in virulence when obtained from different sources. In the saliva of healthy persons, which seems to be its normal habitat, it sometimes has comparatively little virulence. On the other hand, when contained in the blood or in an exudate from a serous cavity of an infected rabbit or mouse, it is very virulent. In one instance (1881) the writer has seen a fatal result in a dog from the subcutaneous injection of 1 c.c. of bloody serum from the subcutaneous connective tissue of a rabbit recently dead. Pneumonia never results from subcutaneous injections into susceptible animals, but injections through the thoracic walls into the lung may induce a typical fibrin- ous pneumonia. This was first demonstrated by Tala- mon (1883), who injected the fibrinous exudate of croup- ous pneumonia, obtained after death, or drawn during life by means of a Pravaz syringe, from the hepatized portion of the lung, into the lungs of rabbits. Gameleia has also induced pneumonia in a large number of rabbits, and also in dogs and sheep, by injections directly into the iDulmonary tissue. Sheep were found to survive 218 IMMUNITY AND SERUM-THERAPY. subcutaneous inoculations, unless very large doses (5 c.c.) of a virulent culture were injected. But intrai^ulmonary inoculations are said to have invariably produced a typi- cal fibrinous pneumonia which usually proved fatal. In dogs similar injections gave rise to a " frank, fibrinous pneumonia which rarely proved fatal, recovery usually occurring in from ten to fifteen days, after the animal had passed through the stages of red and gray hepatiza- tion characteristic of this affection in man." Without doubt an attack of pneumonia is followed by a certain degree of immunity of longer or shorter dura- tion. According to Huge, who has recently made a care- ful study of the subject, relapses are very infrequent — in- dicating a temporary immunity — but subsequent attacks are more likely to occur in those who have once suffered an attack of the disease, and as many as four or five at- tacks have been known to occur in the same individual. In 1,100 cases collected by Wagner but two relapses occurred (= 0.18 per cent.). Huge reports that in 440 cases treated at the Charite in Berlin there were but two relapses. The liability to subsequent attacks at a later period is shown by the following figures, which we copy from Kuge's paper: In 280 cases reported by Stortz, 26.4 per cent, had previously suffered an attack of the disease ; in 133 cases reported by Morhart the jaropor- tion of previous attacks was 41.3 per cent. ; in 157 cases by Pohlmann, 84.4 per cent. ; in 166 cases by Schapira, 31.3 per cent. ; in 128 cases by Keller, 36.9 per cent. : in 175 cases by Grisolle, 30.9 per cent. The writer, in a series of experiments made during the winter of 1880-81, obtained experimental evidence which showed that susceptible animals (rabbits) acquire im- munity from the pathogenic action of this micrococcus PNEUMONIA. 219 as a result of inoculations with an attenuated virus. The experiments referred to had as their object the determi- nation of the comparative vahie of various germicidal ag-ents, as tested upon this micrococcus ; incidentally it was found " that a protective intiuence has been shown to result from the injection " (into rabbits) " of virus, the virulence of which has been modified, without being- en- tirely destroyed, by the agent used as a disinfectant." (Quoted from the writer's report of the experiments re- ferred to, Johns Hopkins University, " Studies from Biological Laboratory," Baltimore, 1882.) In 1891 G. and F. Klemperer published an important memoir relating to the pathogenic action of this micro- coccus and the production of immunity in susceptible animals by means of filtered cultures. In some cases this immunity was found to last as long as six months. A curious fact developed in their researches was that the potency of the substance contained in the filtered cult- ures was increased by subjecting these to a temperature of 41° to 42° C. for three or four days, or to a higher temperature (60° C.) for an hour or two. When injected into a vein after being subjected to such a temperature, immunity was complete at the end of three or four days ; but the same material, not so heated, required larger doses and a considerably longer time (fourteen days) to confer immunity upon a susceptible animal. The un- warmed material caused a considerable elevation of tem- perature, lasting for some days. The authors mentioned ccmclude from their investigations that the toxic sub- stance present in cultures of Micrococcus pneumoniae crouposse is a proteid substance, which they propose to call pneumotoxin. The substance produced in the body of an immune animal, as a result of protective inocula- 220 IMMUNITY AND SERUM-THEEAPY. tions, upon wliicli the immunity of these animals de- pends, is also a jsroteid, which they call antipneumotoxiu. This they isolated from the blood-serum of immune ani- mals. By exx)eriment the}' were able to demonstrate that the blood-serum containing this pr^otective proteid, when injected into other animals, rendered them im- mune ; and also that it arrested the progress of the in- fectious malady indiiced by inoculating susceptible ani- mals with virulent cultures of the micrococcus, ^'hen injected into the circulation of an infected animal its curative action was manifested by a considerable reduc- tion of the body temperature. The toxalbumin was ob- tained from filtered bouillon cultures of a virulent va- riety of the micrococcus of pneumonia, in the form of an amorphous yellowish-white powder. This was thrown down from the filtered cultures by means of alcohol, and again dissolved in water and reprecipitated in order to purify it. Issaeff (1893) as a result of his experiments has found that the virulence of this micrococcus can be greatly in- creased by successive inoculations in the peritoneal cav- ity of rabbits, and that after a series of ten or twelve such inoculations the blood of the infected animal does not coagulate and becomes extremeh' toxic. In order to obtain the toxins from this blood Issaeff collects the blood of three or four animals just dead in a sterilized vessel, and adds to this an equal volume of sterilized water containing one per cent, of glycerin, made alka- line by the addition of a few drops of a concentrated solu- tion of bicarbonate of soda. The mixture is sterilized by passing it through a Chamberland filter. This liquid sometimes kills rabbits when injected into the circiilation in the proportion of one per cent, of the weight of the PNEUMONIA. 221 animal. When heated to 70° C. its toxic power is consid- erably diminished and a temperature of 100° C. neutral- izes it completely. Emmerich (1891) has succeeded in immunizing' rabbits and mice by the intravenous injection of a very much di- luted but virulent culture of the micrococcus. Other rab- bits and mice were rendered immune by injecting into them material obtained from rabbits immunized with diluted cultures. The flesh of these animals was rubbed up into a pulp, and the juices obtained by pressure through a piece of sterilized cloth. The bloody juice, after standing for twelve hours at a temperature of 10° C, was passed throug-h a Pasteur Alter and then served to immunize the animals referred to. Belfanti (1892) has succeeded in immunizing rabbits against the pathogenic action of this micrococcus by in- jecting" into the circulation a filtrate obtained from the sputa of pneumonia cases. The viscid sputa mixed with an equal part of distilled water was kept on ice for twenty-four hours and then passed through a Chamber- land filter. Ten c.c. of this filtrate was injected into the ear vein of rabbits. Some of the animals so treated proved to be immune against general infection when in- oculated with a virulent culture of the micrococcus, but they had a localized inflammation and oedema about the point of inoculation. After recovering from this they proved to be entirely refractory against subsequent in- oculations. Foa and Scabia (1892) have reported success in produc- ing immunity with filtered cultures, and also with a glyc- erine extract from the blood of an infected rabbit. This, after filtration, was injected subcutaneously in doses of 2 c.c. at intervals of five days. The authors named 222 IMMUNITY AND SERUM-THERAPY. have also produced immunity in rabbits by the use of "pneumo-protein." This is an extract from the bacterial cells obtained by first collecting- these from the surface of a Chamberland filter throug-h which the cultures have been passed ; then dig-esting- them for three hours at 55° C in a five per cent, solution of g-lycerin. According- to Foa and Scabia immunity produced in this way is more decided and of long-er duration than that resulting- from the other methods tested by them. Mosny (1892) has also made numerous experiments which show that rabbits may be immunized by means of filtered cultures, or by the juices from the tissues of an immune animal obtained by maceration and filtration. When sterilized cultures were employed the best results were obtained by first heating- very virulent cultures for three hours at 60° C. The dose employed was 10 c.c, and immunity was not established immediately bat required a period of at least four days for its development. The blood-serum of immune rabbits was not found to have any bactericidal power, and the micrococcus of pneu- monia preserved its vitality longer in the blood-serum of immune rabbits than in that of other animals of the same species. G. and F. Klemperer had previously reported that the blood of immune rabbits does not destroy the micrococ- cus of pneumonia or restrict its development. Issaeflf (1893) also reports his success in immuniziug- rabbits by means of sterilized cultures or filtered blood from infected animals recently dead. A single intrave- nous injection of 10 c.c. of filtered blood, prepared as here- tofore indicated (p. 220) suflficed to confer immunity. To test immunity the animals were subsequently inoculated with two to four drops of virulent blood ; and to maintain PJTEUMONIA. 223 it the inoculatious (0.5 c.c.) were repeated every four weeks. Although immune against infection these ani- mals are said not to have acquired any immunity against the toxins of the micrococcus of pneumonia. Contrary to the conclusion reached by G. and F. Klemperer, Is- saeff concludes from his experiments that " rabbits, although completely refractory against pneumonic infec- tion, remain highly sensitive to the toxins of this mi- crobe. Even small doses of the toxins are not neutralized in the blood of vaccinated animals. We are therefore brought to the conclusion that the existence of an anti- toxic property of the blood of vaccinated animals cannot be admitted." The serum of immunized rabbits was not found by Issa- eff to possess any bactericidal power for the micrococcus of pneumonia, and no attenuation of virulence occurred as a result of cultivation in this serum. But when intro- duced beneath the skin of an immune rabbit the micro- coccus quickly loses its virulence. At the end of eigh- teen hours it has completely lost its pathogenic power, and cultures made in bouillon no longer have any injuri- ous effect upon rabbits. This attenuating effect pro- duced in the body of an immune animal is ascribed by Issaeff to the action of phagocytes, which are said to be very numerous, and in the course of five or six hours to pick up all of the cocci in the vicinity of the point of inoculation. These are not, however, immediately de- stroyed in the interior of the phagocytes, but preserve their vitality for nearly forty-eight hoiirs, and when intro- duced into bouillon give a culture which has no longer any pathogenic virulence. 224 IMMUNITY AND SERUM-THERAPY. Serum-therapy. G. and F. Klemperer (1891)- having- succeeded in im- munizing' rabbits against infection by this micrococcus IDi'oceeded to experiment with the blood-serum of im- mune rabbits as a therapeutic agent. They were suc- cessful in saving infected rabbits by the intravenous injection of such blood-serum in the dose of 8 c.c. ad- ministered as late as twenty-four hours after the subcu- taneous injection of a virulent culture. Subcutaneous injections were less effective, and often failed after an interval of twenty -four hours from the time of infection, but were successful when g-iven within eight or ten hours. The tissue juices of immune rabbits also proved to have a curative power. They then proceeded to test the therapeutic value of immune rabbit's serum on man by injecting, in six cases, from 4 to 6 c.c. beneath the skin. They report that in every case a notable fall of temperature followed the in- jection. In two cases it remained normal, and in two again mounted to the former point at the end of six hours. The authors referred to recognized the fact that further experiments would be required to establish the value of the treatment. Mosny (1892) was not able to confirm the results of G. and F. Klemperer as to the therapeutic value of blood- serum or tissue juices of immune animals when injected into rabbits after infection, even when this was done after a very short interval. On the other hand, Foa and Scabia (1892) succeeded in curing infected mice and rabbits by the subcutaneous in- jection of blood-serum from immune animals. But in ten cases of pneumonia in man, treated with doses of 5 to 7 PNEUMOl^IA. 22o c.c. of blood-serum from immune rabbits, their results were less favorable tliau those reported by the Klem- perers. The dose mentioned was repeated two or three times at intervals of two days. In six of the ten cases no modification of the course of the disease occurred, and the crisis took place on the ninth or tenth day. In the other four cases the crisis occurred in from twenty-four to forty-eight hours after the first injection, but it would of course not be safe to conclude that this was a result of the dose given. Emmerich (1894), referring to the experiments made by Klemperer and by Foa, says that the rabbits from which they obtained their serum were not completely immu- nized. In his own experiments such a degree of immu- nity was acquired that the animals resisted doses of 30 to 40 c.c. of a virulent culture. This immunity did not, ac- cording to Emmerich, depend upon the presence of an antitoxin in the blood of the immune animal, but upon the destruction of the micrococci when introduced into the body of the animal by some bactericidal substance present in the blood. Emmerich and Fowitzky have given the results of their experiments in serum-therapy in a paper published in 1891. These were not numerous, but indicated that the blood-serum of rabbits immunized by their method has the j)ower of curing the acute infec- tious disease (septicaemia) produced in susceptible ani- mals (rabbits and mice) by inoculation with a culture of the micrococcus of croupous pneumonia. In Emmerich's last published paper upon the subject he gives no addi- tional experimental evidence but says : " That the active substance [Heilstoffe) in the serum of immune animals will also some time prove to have an 15 226 IMMUNITY AND SERUM-TITERAPY. ideal curative power for man is beyond doubt and only a question of time." BIBLIOGRAPHY. Arkharow, J. : Recberches sur la guerisou de 1' infection pneumoniquc cbez les lapius, etc. Arch, de Med. Experim. et d'Anat. Pathol., iv., 4. Belfanti, S. : Sulla imraunizatione del coniglio per mezzo dei filtrati di spiito pueumouico. Riforma Med. , 1892, No. 126. Bunzl-Federu : Ueber Immunisierung und Heilung bei der Pneumo- kokken-Infektiou. Arch, t'tir Hygiene, 1894, Bd. XX., p. 152. Emmerich, Rudolf : Ueber die Infection, Immuuisirung und Heilung bei crouposer Pueumonie. Zeitschr. ftir Hyg., XVII., 167, 1894. Emmerich und Fowitzky : Die ktinatliche Erzeugung von Immunitat gegen croupose Pneumonic und die Heilung dieser Krankheit. Miinch. med. Wchscbr., 1891, No. 32. Foa, P., e Scabia, E. : Sulla Pueumoproteina. Gazz. Med. di Torino, 1892, p. 421. Issaeflf, B. : Contribution a I'etude de I'immunite acquise contre le pneumocoque. Ann. de I'lnst. Pasteur, 1893, p. 260. Klemperer, G. und F. : Versuche uber Immuuisirung und Heilung bei der Pneumokokkenlnfektion. Berl. klin. Wchschr., 1891, Nos. 84 and 35. Klemperer, G. : Klinischer Bericht fiber einige Falle von specifisch be- haudelter Pueumonie. Centralbl. f. klin. Med., 1892, p. 40. Ruge, H. : Ueber das Pneumonie-Recidive. Charite-Annalen, Bd. XIX., 1894, p. 184. Pane, N. : Richerche sull' immunizzazione del conigli contro il bacillo • setticoemico dello sputo mediante del batterio virulento. Riv. Clin. eTerapeut., 1892, p. 641. Ripristinamento della virulenza del diplo-bacillo pneumonioe mediante il virus carbonchioso. Riforma Med., 1893, p. 147. Mosny, E. : Action sur le pneumocoque du serum sanguin des lapins vaccines contre I'infection pneumonique. La Semaine Med., 1892, p. 98. Sur la vaccination contre I'infection pneumonique. Arch, de Med. exper. et d'Anat. pathol., iv., 195, 1892. xiy. RINDERPEST. The disease of cattle known in Germany as rinderpest is due to a bacillus closely resembling- the bacillus of fowl cholera and of swine plague {Bacillus septiccemice hemorrhaglcce). Professor Semmer, of St. Petersburg, has reported (1892) his success in immunizing cattle against this dis- ease. The virulence of cultures was attenuated by pass- ing them through guinea-pigs, or by exposure to heat, and this attenuated virus was used in protective inocu- lations. Semmer says : " By the subcutaneous injection of blood-serum from immune animals their susceptibility to rinderpest was diminished, and such blood-serum destroyed the ' rinder- pest contagium ' in one to twenty -four hours." BIBLIOGRAPHY. Semmer, E. : Ueber das Rinderpestcontagium und tiber Iramunisierung und Schutzimpfung gegen Rinderpest. Berl. tierarztl. Wochen- schr., 1893, 590. XV. SMALL-POX. Inoculations with virus obtained from a pustule on a small-pox patient were extensively iDractised before the discovery of vaccination by Jenuer. These inoculations o-ave rise to a mild attack of the disease, followed by im- munity which was apparently as complete as that follow- ing a more severe attack contracted in the usual way. This method seems to have been practised by Eastern nations long- before it was introduced into Europe. It was extensively employed in Turkey early in the eigh- teenth century, and was introduced into England through the influence of Lady Mary Wortley Montagu. No doubt the mortality from small-pox was greatly diminished by these inoculations, but they were attended by the disad- vantage that the disease was propagated by them, in- asmuch as inoculated individuals became a source of in- fection for others. Inoculation was still practised in England for some time after the demonstration of the protective value of vaccination, but in 1840 it was pro- hibited by an act of Parliament. The usual course of the disease as a result of inoculation was as follows : On the second day a papule developed at the point of inocu- lation, which became vesicular on the fourth day, and pustular on the eighth, at which time the patient had a chill, followed by fever and tumefaction of the axillary SMALL-POX. 229 glands. This was followed by the ordinary small-pox eruption as seen in a mild case. There is some evidence that vaccination as a protection against small-pox was practised to a limited extent prior to the time of Jenner. Thus Von Humboldt has stated that it was known at an early period to the Mexicans. But its introduction as a reliable method of protecting against smallpox is due to the patient researches of the renowned English physician, whose attention was first attracted to the subject in 1768, although it was not until 1796 that he made his first vaccination in the human sub- ject. His first public institution for the practice of vac- cination was established in 1799, and the following year the practice was introduced into France, Germany, and the United States. Vaccinia in the cow was more frequent before the in- troduction of vaccination than at present, and often pre- vailed as a veritable epidemic. This was no doubt due to the greater prevalence of small-pox, and also to the fact that milkers, not protected by vaccination, by the sores on their hands, arising from contact with the teats of an infected cow, communicated the disease to other animals. We cannot attempt to review the evidence for and against the view that vaccinia in the cow is simply a modified form of small-pox, rather than a specific infec- tious disease of the bovine species, as has been claimed by numerous physicians of prominence. We believe, however, that the former view is supported by experi- mental evidence, as well as by the analogy resulting from recent additions to our knowledge relating to pro- tective inoculations in other infectious diseases. Nor is it necessary to devote any space to the methods 230 IMMUNITY AND SERUM-TIIERAPY. of propagating vaccine virus, and the operation of vac- cinating-, with its immediate results. But some account of the value and duration of the immunity conferred by a successful vaccination seems desirable. In the early days of vaccination it was claimed that a successful vaccination would confer a life-long immunity against small-pox ; but this has been disapproved by ample experience, and it is now generally recognized that the immunity usually expires after a time, which varies greatly in different individuals, and can only be determined for each by a revaccination practised at proper intervals, or when small-pox is prevalent. The fact that a single attack of small-pox is not alwa3's pro- tective would lead us to expect that the immunity from vaccination would not be absolute, and experience shows that in every small-pox epidemic a certain number of persons who have been vaccinated fall victims to the disease ; but experience also shows that the mortality among the vaccinated is very much less than among the non-vaccinated. The London Board of Health, from thirty returns sent to them — not selected — has given the following table : Natural small-pox in the un- protected Small-pox after small-pox .... • Small-pox after vaccination.. Number of Cases. 1,731 58 929 Deaths. 361 22 32 Percentage of Deaths. 20.85 37.92 3.44 The gradual loss of immunity from vaccination, as determined by revaccination, is shown by the following figures, which are given in a recent (1894) paper by Bie- SMALL-POX. 231 dert. In the year 1889 small-pox was introduced into Hag-enau, and twenty-one cases occurred. The physician officiall}^ charg-ed with that duty revaccinated all of the sOhool-children with the result ffiven below : Age. Number Vaccinated. Successful. Per Cent. 6 to 7 288 292 322 221 306 413 94 187 Wl 170 273 307 33 7 to 8 63 8 8 to 9 72 5 9 to 10 80 10 to 11 85 8 11 to 12 88.6 We may remark in connection with this table that in Germany vaccination is compulsory, and all children must be vaccinated before the September of the year follow- ing- their birth. All scholars in public or private schools who have not had an attack of small-pox must be revac- cinated when twelve years old. The experimental evidence presented in the section on acquired immunity (Part First, Section II.) sug-g-ests the possibility that in small-pox and other infectious dis- eases, in which the specific infectious agent has not yet been demonstrated, the immunity resulting from an at- tack — or in small-pox from vaccination — may also be due to the presence of an antitoxin in the blood of the im- mune individual. The writer undertook some experiments with a view to determine this question in the spring of 1892, but owing- to various causes has never been able to complete the in- vestigation. The experiments made, however, indicate that the blood of a recently vaccinated calf contains an antitoxin which neutralizes the potency of vaccine virus, either bovine or humanized. The exjaeriments are re- 232 IMMUIilTY AND SERUM-THERAPY. corded in my paper on the " Practical Results of Bacteri- ological Researches " as follows : These experiments were made with the kind assistance of Dr. Wm. E. Griffiths, of Brooklyn, who has for many years been engaged in the production of vaccine virus, and consequently is an expert in the vaccination of calves and in recognizing vaccinia in these animals. "Upon visiting Dr. Griffiths and making known to him my desires, I found him quite willing to assist me, and also that he had a recently vaccinated, and conse- quently immune, calf in his stable. This animal had been vaccinated in numerous places upon the abdomen and thighs fourteen days previously. The vaccination was entirely successful, and a large number of quills had been charged from the vesicles which formed. At the time of my visit for the purpose of collecting blood-serum from this animal, dry crusts still remained attached at the points where vaccination had been practised two weeks previously. On the 28tli of April I collected blood-serum from a superficial vein in the hind leg of this calf. This blood was i^laced in an ice-chest for twenty -four hours, at the end of which time the clear serum was drawn off in ' Sternberg's bulbs.' Four drops of this serum were placed in each of two small, sterilized, glass tubes ; in one of these we placed three quills charged with fresh vaccine lymph from a calf. At the end of an hour the quills were removed, after carefully washing off in the serum the lymjjh with which they had been charged. In the other tube we mixed with the four drops of blood- serum an emulsion made from a fragment of a perfectly fresh vaccine crust from the arm of a child ; this was crushed upon a piece of glass and rubbed up with a little of the same blood-serum. The two tubes were now placed in an ice-chest for twenty-four hours, at the end of which time the contents were used to vaccinate a calf purchased for the purpose. Dr. Griffitlis carefully shaved the thiffhs of this calf and scarified each thiffh in several SMALL-POX. 233 places, as he is accustomed to do in vaccinating- for the propagation of lymph. The contents of the tube con- taining- lymph from the quills was rubbed into the scari- fied places upon one thig-h, and .the contents of the tube containing- the emulsified crust into the other. On the 8th of May, nine days after the vaccination, the calf was carefully examined, and it was ascertained that the result of the vaccination was entirely negative. " Evidently it was necessary to make a control experi- ment before we would be justified in ascribing- this neg-a- tive result to a neutralization of the virus by some special substance present in the blood-serum of an immune calf. Possibly the blood of a non-immune calf might also, after an exposure of twenty-four hours, neutralize the specific virulence of vaccine lymph. The control experi- ment was made as follows : "On the 9th day of May we collected blood from a vein in the leg- of a non-immune (not vaccinated) calf ; tliis was placed in the ice-chest for twenty-four hours, and the following- day clear serum was collected in Sternberg's bulbs. Three quills, charged with fresh lymph from a calf, of the same lot as those used in the previous experi- ment, were placed in four drops of this blood-serum in each of two small glass tubes. As in the previous ex- periment, the lymph was washed from the quills at the end of an hour, and the tubes were placed aside in the ice-chest. At the end of twenty-four hours the serum in these two tubes was used to vaccinate the same calf which had served for the previous experiment Several points were scarified upon the left thigh and upon the left side of the abdomen, which were carefully shaved for the pur- pose. "At the same time the animal was vaccinated upon the right thigh and upon the right side of the abdomen with virus mixed with the blood-serum from the immune calf. This serum, collected in Sternberg's bulbs on the 28th of April, had since been kept in the ice-chest. One hour be- fore the vaccination four drops of this blood-serum were 234 IMMUNITY AND SERUM-TIIERAPY. mixed with one drop of liquid lymph, which had been re- cently collected by Dr. Griffiths in a cai^illary tube from a vaccinated calf. At the same time three quills charged with bovine lymph were immersed in four drops of the same blood-serum — from immune calf. As stated, the animal was vaccinated upon the right side of the ab- domen and upon the right thigh with this virus, which had been exposed for one hour to the action of blood- serum from an immune calf. The serum containing the liquid lymph was rubbed into the scarification on the right side of the abdomen, the serum containing lymph from the quills into the right thigh. On the 19th of May, eight days after the vaccination, the animal was carefully examined by Dr. Griffiths and myself, and the following results noted : Upon the left thigh and left side of the abdomen the vaccinations — from quills in non- immune blood-serum after twenty-four hours' contact — were entirelj^ successful, the scarification being sur- rounded by characteristic vesicles and covered by char- acteristic crusts. Upon the right thigh — vaccinations from quills immersed in blood-serum from immune calf for one hour — and upon the right side of abdomen — vac- cinations with liquid lymph mixed with blood-serum from immune calf — the result was entirely negative. Several of the scarifications had entirely healed ; others were covered with a dry scab which was easily detached and under which the scarification was healing without any appearance of vesicles such as surrounded the scari- fications upon the left side." * Later I made a number of experiments upon unvacci- nated children in two orphan asylums in Brooklyn, with a view to ascertain whether blood-serum from an immune calf, or from an individual who had recently sufi'ered an attack of small-pox, if injected into th6 subcutaneous * These results have since been confirmed by Kinyon. SMALL-POX. 235 tissues at the time of vaccination, would prevent the de- velopment of a characteristic vaccine vesicle. In these experiments from 1 to 5 c.c. of the serum supposed to contain an antitoxin of small-pox was injected near the l)oint of vaccination, or, in some instances, into the other arm. The result was negative, even when serum was used from an individual who was just convalescent from a severe attack of small -pox. But it may be that a dif- ferent result would have been obtained if a larger quan- tity of blood-serum had been used, or if it had been in- jected into the circulation instead of into the subcuta- neous tissues. More recent experiments by Kramer and Boyce (1893) and by Landmann (1894) also show that small amounts of serum from immune calves (5 to 10 c.c.) do not prevent the development of the vaccine vesicle ; and that blood-serum (25 c.c.) from one who had suffered a recent attack of small-pox did not have any noticeable effect upon the development of a conflu- ent case of small-pox in a child five years of age (Land- mann). Tliis line of research, which appears to us well worthy of attention, is still oi)en for investigators. Immune calves may be obtained at any vaccine establishment, and it is a simple matter to collect sterile blood-serum in consider- able amounts from these immune animals. We have been anxious to see a test made as reg-ards the possible thera- peutic value of such serum. It could be injected into the circulation of a small-pox patient through the median vein, and there is no reason to suppose that there would be any danger in introducing- a considerable quantity in this way. As the writer is not likely to have an oppor- tunity to follow up these experiments he takes this occa- sion for inviting- the attention of others, more favorably 236 IMMUNITY AND SERUM-THERAPY. situated for such an investigation, to this promising field for experimental work.* BIBLIOGRAPHY. Anche : Essai de serotherapie dans la variole. Arch. clin. de Bordeaux, 1893, vol. ii., p. 317. Kinyon : Preliminary report on the Treatment of Variola by its Anti- toxin. Abstract of Sanitary Reports, Marine Hospital Service, vol. X., p. 31, 1894. Kramer and Boyce : The Nature of Vaccine Immunity. British !Medi- cal Journal, Xoveniber, 1893. Laudmauu : Fiuden sich Schutzstoffe in dem Blutserum von Individuen, welche Variola bezw. Vaccine iiberstanden haben ? Zeitscbr. ftlr Hygiene, Bd. XVIII., 1894, p. 318. Ruete, A., und Enoch, C. : Ueber Vaccinereinkulturen und fiber dus toxin " Vaccinin." Dtsche. med. Wohschr., 1893, p. 547. Sternberg : Practical Results of Bacteriological Researches. Transac- tions of the Association of American Physicians, 1892, p. 98. * Since the above was written, Kinyon has reported two cases in which blood- serum from an immune calf was injected subcutaneously in small-pox patients in doses of 15 to 30 c. c. with supposed good results in one. • XVI. SWINE PLAGUE. As stated in the chapter on cholera in fowls, the bacil- lus of SAvine plag'ue {Schwelneseuche, Loffler and Schiitz) very closely resembles Pasteur's microbe of fowl cholera and Koch's bacillus of rabbit septicaemia, and if not iden- tical with these at least varies from them so slightly in its morphological and biological characters that recent authors do not feel justified in considering it a distinct species. Koch first obtained his bacillus of rabbit septi- ciemia by inoculating rabbits with putrefying flesh infu- sion. Gaffky produced the same infectious disease in rabbits by inoculating them with impure river-water. Davaine had previously obtained similar results by inoc- ulating rabbits with putrefying blood. The writer in 1887 produced the same disease in rabbits, while in Cuba, by inoculating them with putrefying liver from a yellow fever cadaver. A similar, and possibly identical, bacillus has been found in the blood of deer (Hueppe), of cattle (Kitt), and of buffalo (Oreste - Armanni) suffering from a fatal infectious disease. And all of these allied species or varieties are included by Huepi^e and by the present writer under the single specific name Bacillus septiaemife liemorrhagicce. The bacillus of the disease known in this country as swine plague, according to Smith, agrees in all particulars with that of the German swine plague {Schweineseuche) described by Lofiler and 238 IMMUNITY AXD SEKUM-THEUAPY. Schiitz, except that tlie latter is more pathogenic for swine and for rabbits. • In a recent publication Smith and Moore (United States Department of Ag-riculture, Bureau of Animal In- dustry, Bulletin No. 6, 1894:) have given an account of their experiments relating to immunizing animals against the pathogenic action of this bacillus. The bacilli used in these experiments were sufficiently virulent to kill rabbits in twenty hours when injected beneath the skin of these animals in doses of 0.001 c.c. of a fresh bouillon culture. The experiments were made upon young adult rabbits by various methods, viz. : with sterilized bouillon cultures ; with sterilized suspensions of agar cultures ; with the filtrate of agar suspensions ; with defibrinated, sterilized blood of infected rabbits; with blood-serum from immune animals. " A greater or less degree of immunity was produced in rabbits by sterilized bouillon cultures, sterilized agar suspensions, sterilized blood from infected rabbits, and blood-serum from immunized rabbits. The sterilized blood of diseased rabbits was capable of producing im- munity, while the blood-serum of immune rabbits pro- duced rather equivocal results." The different degrees of immunity which may be ac- quired by rabbits, as shown by a subsequent inoculation with virulent material, are classified by Moore as follows : 1. No resistance — acute sei^ticfemia. 2. Slight resistance — peritonitis. 3. Increased resistance — pleuritis and pericarditis with or without secondary laneumonia. 4. Higher degree of resistance — pleuritis and peritoni- tis. SWINE PLAGUE. 239 5. Still greater resistance — irregular lesions in the form of abscesses, subcutaneous and subperitoneal, 6. Nearly complete immunity — very slight reaction at the point of inoculation. Uj5 to the present time the bacteriolog-ical experts of the Department of Ag-riculture have not proposed to make a practical application of the facts developed in their experimental work in the way of protecting- herds of swine by means of inoculations with an attenuated virus, or with sterilized cultures. In the reijort on swine plague, made by the Bureau of Animal Industry, pub- lished in 1891, the following measures for arresting an epidemic are recommended : " When the disease has actually appeared in a herd the question generally arises whether it is worth while to make any attempt to save a portion of the herd or to leave them to their fate. As a rule it may be stated that it is best to slaughter both healthy and diseased at once, and give the surroundings sufficient time to rid them- selves of the infection before fresh animals are brought into them. If this be not desirable, we should recom- mend the following measures to be rigorously carried out : " a. Removal of still healthy animals to uninfected grounds or pens as soon as possible. " h. Destruction of all diseased animals. " c. Careful burial or burning of carcases. " d. Repeated thorough disinfection of the infected premises. " e. Great cleanliness both as to surroundings and as regards food." In the same report (1891) the following reference is made to protective inoculations : " As regards swine i)lague the experiments which have thus far been carried out indicate that this disease may 240 IMMUNITY AND SEIU'M-TIIEUAPY. prove amenable to preventive inoculation. We have been able, by the injection of both living- cultures and those sterilized at a low temperature (58° C.) to make the most susceptible animals — rabbits — insusceptible to the most virulent swine-plag'ue bacteria. By two subcutane- ous injections of cultures of swine-plag'ue bacteria, swine have been made insusceptible to doses injected into the circulation which proved fatal to control pigs within twenty-four hours." According to Smith the experiments of Metchnikoff (1892), reported as made with the bacillus of hog cholera, were in fact made with the bacillus of swine plague ; we therefore refer to them here. These experiments showed that rabbits could be easily immunized against the patho- genic action of virulent cultures by means of blood, from an infected animal, sterilized by heat. Doses of 1.5 c.c, or more, were fatal to rabbits ; but smallef doses, re- peated several times, given either subcutaneously or by injection into the circulation, caused the animal to be- come immune. BIBLIOGRAPHY. Smith : Special Report on the Cause and Prevention of Swine Plague. United States Department of Agriculture, Bureau of Animal In- dustry, Washington, 1891. Smith and Moore : Op. cit., p. 238. XVII. STREPTOCOCCUS INFECTION. It is now generally recognized by pathologists that erysipelas, puerperal fever, certain forms of diphtheritic inflammation of the fauces, and certain acute abscesses are due to infection by a streptoccus described by recent authors under the name of Streptococcus pyogenes. This streptococcus, like other pathogenic microorganisms of the same class, varies greatly in its pathogenic power as a result of conditions relating to the source of the par- ticular variety under cultivation. As obtained from a case of erysipelas or puerperal fever it is extremely vir- ulent, but when it has led a saprophytic existence for some time, or has been cultivated for a considerable time in the usual artificial culture media, its pathogenic po- tency is greatly diminished. Mironoff (1893) has made a series of experiments with a view to determining whether rabbits can be immu- nized against the pathogenic action of this streptococcus, and has obtained successful results by the following method : Vigorous rabbits, weighing 2 kilogm., were inoculated subcutaneously with from 3 to 6 c.c. of a sterilized bouil- lon culture of the streptococcus. Cultures three days old were employed, and these were sterilized for twenty minutes at 120° C— the reason for using so high a tem- 16 242 IMMUNITY AND SERUM-THERAPy. perature is not apparent, inasmuch as this streptococcus is destroyed in a few minutes by a temperature of 60° C. At the end of ten to fifteen days " when the animal has fully recovered," a second dose of from 6 to 12 c.c. of a culture, sterilized in the same waj', is injected beneath the skin. After another interval of ten to fifteen days 2 c.c. of a virulent non-sterilized culture is injected subcu- taneously, and this is repeated with gradually' increasing doses (1 to 2 c.c. more) at intervals of the same period. Finally the animals " support without reaction " a dose five times as great as would be required to kill an animal of the same weight not immunized. But the author adds that more than half the animals thus treated died before the completion of the immunizing process. These deaths resulted from local infectious processes, such as peritonitis, pericarditis, meningitis, or abscesses formed at the point of inoculation. Further experiments showed that the blood-serum of animals immunized in this way when injected into sus- ceptible animals (rabbits) in the dose of 1.5 c.c. per kilo- gm. of body-weight conferred upon them a certain de- gree of immunity against streptococcus infection, and with twice this amount (3 c.c.) a very decided immunity was produced. The blood-serum of immune rabbits in doses of 3 to 4 c.c. per kilogm. of body-weight was found to exercise a curative power, and to completely ar- rest the acute septicaemia resulting from inoculations with a virulent culture of this streptococcus, or to cause the disease to run a chronic course, with formation of abscesses and final recovery. In this connection we may call attention to the experi- ments of Emmerich (1886), which show that the fatal course of anthrax infection, in rabbits, may be arrested by STREPTOCOCCUS INFECTION". 243 tlie subcutaneous or intravenous injection of this strep- tococcus. Subsequent experiments by Emmerich and de Mattei (1887) showed that eleven hours after such an injection the anthrax bacilli were all dead and were al- ready undero'oing degenerative changes. In a recent communication (1894) Emmerich and his associates have reported numerous additional experi- ments which show that the blood-serum of a rabbit which is suffering- from streptococcus septicaemia (third day), when filtered through a Pasteur-Chamberland filter to remove all living cocci, may be used with success in ar- resting anthrax infection in rabbits. The filtered serum was given four hours after the anthrax infection in the dose of 25 c.c. in the peritoneal cavity and 15 c.c. subcu- taneously. This was repeated the following day at nine o'clock in the morning and five o'clock in the evening, and again on the third day in the morning. Favorable results were also obtained by using in the same way blood-serum from a sheep infected with the streptococ- cus. Cobbett (1894) reports success in immunizing rabbits by means of attenuated varieties of the streptococcus or by filtered cultures. Also that cutaneous erysipelas, pro- duced by inoculation, after recovery leaves the patient immune from a repetition of the local inflammatory proc- ess as a result of a subsequent inoculation, and also con- fers a general immunity against streptococcus infection. But this immunity is of short duration, not lasting long- er than a few weeks. Inoculation in the ear of a rabbit, protected by a previous inoculation in the same locality, is followed by an inflammatory reaction ; but this is of brief duration and has disappeared before the erysipela- tous inflammation jDroduced in a control is well under way. 244 IMMUNITY AND .SERUM-THERAPY. We may also mention here the experiments of Mircoli (1894) with reference to the immunization of rabbits against the pathogenic action of staphylococci (aureus ?) ; and of Rodet and Courmont (1892), who have studied the chemical products of the staphylococci and have tested their pathogenic action upon dogs and rabbits. BIBLIOGRAPHY. Cobbelt : Journal of Pathology and Bacteriology, vol. iii., p. 88, 1894. Emmerich, R. : Die Heilung desMilzbrandesdurchErysipelserum, etc. Miiuch. med. Wchscbr., 1894. Nos. 28-31, pp. 549, 576, 594, 620. Mironoff : Immunisation de.s lapins contre le streptocoque, etc. Arch, de Med. experim. et d'Anat. pathol., 1893, p. 441. Mircoli, S. : Immunizzazione del coniglio contre la setticemia da startl- ococco piogeno. Gazz. d. Ospit., 1894, p. 203. Rodet, A., et Courmont, J. : fitude experimentale des substances solu- bles toxiques elaborees par le staphylocoque pyogeue. Rev. de Med., 1893, No. 2. xvin. SYMPTOMATIC ANTHRAX. This disease of cattle is popularly known as " black leg-," or " quarter evil," and is described by German authors under the name of Rauschhrand — French, " ehar- boii symptoiiiatiqiLer The disease prevails during- the summer months in various parts of Europe, and to some extent in the United States. It is characterized by the appearance of irregular, enlphysematous swellings of the subcutaneous tissues and muscles, especially over the quarters. The muscles in the affected areas have a dark color and contain a bloody serum in which the bacillus is found to which the disease is due. This is an anaero- bic bacillus which forms large oval spores. The etiolog}^ of the disease was first clearly established by the researches of Arloing, Cornevin, and Thomas (1880 to 1883), and subsequent researches have shown that im- munity may be produced in susceptible animals by pro- tective inoculations. The disease causes considerable losses among cattle in certain sections. Horses do not contract it spontaneously, and when inoculated with a culture of the bacillus present only a limited local reaction. Swine, dogs, rabbits, fowls, and pigeons have but slight susceptibility. The re- searches of the authors above mentioned have shown that the virulence of a culture is greatly increased by adding to it twenty per cent, of lactic acid. The guinea-pig is 246 IMMUNITY AND SERUM-THERAPY. the most susceptible animal and succumbs in from twenty-four to thirty-six hours when inoculated subcu- taneously with a small quantity of a pure culture. Ac- cording- to Kitasato cultures in a bouillon made from the flesh of the guinea-pio: soon lose their virulence, while cultures in solid media preserve their virulence for a long- time. Cultures ai'e readily attenuated by heat, ac- cording- to the method of Toussaiut and Chauveau — ex- posure to a temperature of 42° to 43° C. in the absence of spores. The spores are attenuated by exposure for several hours to a temperature of 80° to 100° C. Arloing, Cornevin, and Thomas recommend for the production of immunity in cattle inoculation with a dried jjowder of the muscles of animals recently dead from the disease. This is attenuated by heat. According to Kitt the mus- cles should first be dried at 32° to 35° C. and then pow- dered. Two " vaccines " are prepared from this powder — a strong vaccine by exposure to a temperature of 85° to 90° C. for six hours, and a weaker vaccine by exposure for the same time to a temperature of 100° to 104° C. (dry heat). An inoculation is first made with the weaker vaccine which gives rise to a local reaction of moderate intensity. Later a second inoculation is made with the stronger vaccine, after which the animal is immune from the pathogenic action of the most virulent material. Im- munity may also be secured by intravenous injections ; or, in guinea-pigs, by inoculations with cultures which have become attenuated by being kept a few days, or by exposure to a temperature of 42° to 43° C. ; or by inocu- lation with a very small quantity of a pure culture ; or by inoculations with filtered cultures (Roux and Chamber- land) ; or with cultures sterilized by heat (Kitasato). A non-fatal and protective local infection may also be pro- SYMPTOMATIC ANTHRAX. 247 ducecT in cattle by inoculatious with virulent material made into the extremity of the tail. Roux has claimed that animals which have an acquired immunity against symptomatic anthrax are also immune against the patho- genic action of the bacillus of malignant oedema; but Kitasato was unable to confirm this. Strebel, in 1885, published the results of jorotective in- oculations made in Switzerland in 1884. The inoculations were made in the end of the tail with two " vaccines," with an interval between the two of from nine to fourteen days. The vaccines were prepared by exposure to heat, as above recommended by Arloing, Cornevin, and Thomas. The most favorable season for inoculations was found to be the spring, and the most favorable age of cattle for inoculation from five months to two years. In seven Swiss cantons 2,199 cattle were inoculated ; 1,810 inoculations were made among animals which were exposed in dangerously infected pastures. Of these but two died, one two months and the other four months after the protectivjB inoculations. Among 908 inoculated cat- tle, which were pastured with 1,650 others not inocu- lated, the mortality was 0.22 per cent., while the loss among the latter was 6,1 per cent. The following year (1885), according to Strebel, the number of inoculations, exclusive of those made in the canton of Bern, was 35,000. The losses among inoculated animals are reported as having been about five times less than among those not protected in this way. In the canton of Bern, in the same year, according to Hess, 15,137 cattle were inocu- lated by thirty-eight veterinarians — 12,190 of these were pastured in dangerously infected pastures. The results are said to have been favorable to the method, but the abstract at hand does not give the precise figures. 248 IMMUNITY AND SI<:UUM-TIIEKAPY. In 1887 Kitt repoi*ted the results of his investigations, which were confirmatory of those previously published by Aiioing-, Cornevin, and Thomas, and also of a new method of inoculation, which presented the advantag-e that a single inoculation was sufficient to confer immu- nity. This was made in the region of the shoulder with a vaccine somewhat stronger than that emjjloyed by the French bacteriologists, but which was found to be with- out danger for cattle. It produced only a slight local effect. His vaccine was prepared by heating the moist- ened flesh of an animal just dead from the disease to 85° to 90° C. for six hours. This did not kill the spores present, but caused a sufficient attenuation in their viru- lence, t In a later communication (1888) Kitt recommends that the flesh of the diseased animal be first dried and pulver- ized, and then subjected to a temperature of 100° C. in streaming steam for six hours, after which it is to be again dried and used for subcutaneous inoculations. The dose is from 5 to 15 ctgr. Roux (1888) has shown by experiment that sterilized cultures of the bacillus, which have been exposed to a temperature of 115° C, when injected in doses of 40 c.c, three times repeated, into the cavit}' of the abdomen of guinea-pigs, cause these animals to be completely im- mune against the most virulent material. Cultures from which the bacilli have been separated b}^ filtration are still more active. And immunity could easily be con- ferred by the subcutaneous inoculation, in guinea-pigs, of 1 c.c. of the filtrate from the serum obtained from the oedematous tissues of a diseased animal. Schuhanka (1888) has reported the results of inocula- tions made in the dukedom of Salzburg during the j^ear SYMPTOMATIC ANTHRAX. 249 1887. In all 2,596 cattle were inoculated once, and 2,472 twice, with an attenuated virus, in forty-seven different parishes. Most of these were from six months to a year old. No losses occurred as a result of the inoculations. During- the summer of 1887 the 2,472 cattle Avhich had been twice inoculated were associated in infected past- ures with 3,561 unprotected cattle. The loss among- the former was 8, = 0.32 per cent., among- the latter it was 235, = 6.31 per cent. Strebel reports similar results, in 1887, in the canton Freiburg-, where 1,725 cattle which had been inoculated suffered a loss of 0.23 per cent., and 1,945 associated cat- tle a loss of 5.28 per cent. Lydtin (1892) reports the results of inoculations made in five districts {Amtsbezirken) in Baden during- the years 1886-91 : 2,797 cattle were inoculated with a loss of three only as a result of the inoculation. None of the inoculated cattle subsequently contracted the dis- ease. In the Bulletin of the Central Society of Veterhiary Med- icine of France (1892) Guillod and Simon give the results of 3,500 inoculations made since 1884. The mortality among cattle in the region where these inoculations were practised had been from ten to twenty per cent., but fell to 0.5 per cent, among the inoculated animals. The authors last named prefer inoculations in the re- gion of the shoulder to the plan first practised of inocu- lating in the end of the tail. Strebel also (1892) advo- cates this method, which is quickly carried out and at- tended with but little loss. According to Strebel the loss among 13,022 inoculated in this way only amounted to five, while the loss among animals inoculated by the old method was twice as great. 250 IMMUNITY AND SERUM-THERAPY. BIBLIOGRAPHY. Arloing, Cornevin et Thomas : Le cliarbou symptomatique du boeuf. 2me ed., Paris, 1887. Moyen de confeier artificielleraent rimmunite coutre le cbarbou symptomatique ou bacterien avec du virus atteuue. Compte-reudu, Acad, des Sci., Paris, xcv., 1883, p. 189. Experiences publiques sur la vaccination du cbarbon symptoma- tique. Arch, vet., Paris, vi., 1881, p. 731. De I'inoculation du charbou symptomatique par rnjection iutra- veineuse, et de I'immunite conferee au veau, au mouton et a la chevre par ce procede. Compte-rendu, Acad, des Sci., Paris, xci.. 1880, p. 734. Guillod et Simon : Du charbon symptomatique ; nouveau precede d'in- oculation. Bull, de la Soc. Ceutr. de Med. veterin., xlvi., 333, 1893. Hess: Bericht iiber die Schutzimpfungeu gegen Rauschbrand, etc., im Kenton Bern wahrend der Jahre 1886-88. Bern, 1889. Kitt, T. : Ueber Rauschbraudschutzimpt'ungen mit Reinkulturen. Mtsch. f. prakt. Tierheilk., 1893, p. 19. Lydtin : Rauschbrandimpfuugen in Baden. Bad. tbieriirtzl. Mittheil- uugen, 1893, Nos. 9 and 10. Roux : Immuuite contre le charbon symptomatique, confere par les substances solubles. Ann. de I'lnst. Pasteur, t. ii., 1888, p. 49. Strebel, M. : Protective Inoculation for Symptomatic Anthrax. Veter- inary Journal, 1893, p. 153. De I'inoculation preservative du charbon symptomatique, etc. Jour, de Med. veter. et Zootech., 1893, p. 11. Rauschbraudsehutzimpfung in der Schultergegend, bzw. a. d. Brustwand. Schweitz. Archiv f. Thierheilk., XXXIV., 526, 1893. Woltf : Schutzimpfungeu gegen Rauschljrand. Berl. Arch, fiir wis- seusch. uud prakt. Thierheilk., 1888, p. 191. XIX. TETANUS. The experiments of Kitasato (1889) show that pure cult- ures of the tetanus bacilhis injected into mice, rabbits, or guinea-pigs produce typical tetanic symptoms and death.* As the presence of this bacillus at the seat of in- jury, in cases of tetanus in man, has now been demon- strated by numerous observers, there is no longer any question that tetanus must be included among the trau- matic infectious diseases, and that the bacillus of Nico- laier and of Kitasato is the specific infectious agent. Kitasato's recently published experiments (1890) show that cultures of the tetanus bacillus which have been sterilized by filtration through porcelain produce the same symptoms, and death, in the animals mentioned, as result from inoculation with cultures containing the ba- cillus. It is evident, therefore, thtU death results from the action of a toxic substance produced by the bacillus. This is further shown by the fact that the bacillus itself cannot be obtained in cultures from the blood or organs of an animal which has succumbed to an experimental in- oculation with an unfiltered culture ; but the blood of an animal killed by such an inoculation contains the tetanus poison, and when injected into a mouse causes its death with tetanic symptoms. 252 IMMUNITY AND SERDM-THEHAPY. When a platinum needle is clipiDed into a pure culture of the tetanus bacillus, and a mouse is inoculated with it subcutaneously, the animal invariably falls sick within twenty-four hours and dies of typical tetanus in two or three days. Rats, g-uinea-pigs, and rabbits are Itilled in the same way by someAvhat larger quantities — 0.3 to 0.5 c.c. (Kitasato). Pigeons are very slightly susceptible. The tetanic symiDtoms are first developed in the vicinity of the point of inoculation ; if the animal is inoculated in the posterior portion of the body the hind legs first show tetanic contraction, if in the forepart of the body the muscles of the neck are first affected. At the autopsy there is a certain amount of hyperaemia at the point of inoculation, but no pus is formed ; in inoculations with garden earth, or accidental inoculations iii man, pus is commonly found in the vicinity of the inoculation wound. The various organs are normal in appearance. Kita- sato says that he has not been able to demonstrate the presence of the bacillus or of spores in the spinal mar- row, the nerves, muscles, spleen, liver, lungs, kidneys, or blood from the heart; nor has he been able to obtain cultures from the various organs. In mice which were inoculated at the root of the tail Kitasato was able to demonstrate the presence of the bacilli at the point of inoculation by the microscopical examination of an ex- cised piece of the tissues for eight to ten hours after the inoculation ; later than this they were not found. In pus from the inoculation wounds of men and animals acci- dentally infected the bacilli are present, but the forma- tion of spores does not always occur. According to Kita- sato, the sooner death has occurred after accidental inoculation the less likely are spores to be found in the rods, but from pus in which no spores are seen cultures TETANUS. 253 of the bacillus may be obtained in which spores will de- velop in the usual manner. Guinea-pig's are even more susceptible to the tetanus poison than mice, and rabbits less so. The amount of filtrate from a slightly alkaline bouillon culture required to kill a mouse is extremely minute — 0.00001 c.c. (Ki- tasato). The tetanic symptoms are developed within three days ; if the animal is not affected within four days it escajjes entirely. The tetanus poison is destroyed by a temperature of 65° C. maintained for five minutes, or 60° for twenty minutes, or 55° for an hour and a half ; in the incubating oven at 37° C. it gradually loses its toxic potency ; in diffuse daylight, also, its toxic power is gradually lost ; in a cool, dark place it retains its orig- inal potency indefinitely ; in direct sunlight it is com- pletely destroyed in from fifteen to eighteen hours ; it is not injured by being largely diluted with distilled water ; it is destroyed in an hour by hydrochloric acid in the proportion of 0.55 per cent. ; terchloride of iodine de- stroys it in the proportion of 0.5 per cent.; cresol in one per cent. — one hour's exposure. In general it is de- stroyed by acids and by alkalies. Blood-serum from cattle, horses, sheep, rabbits, rats, or guinea-pigs does not modify its toxic properties. Brieger (1886) first succeeded in obtaining from impure cultures of the tetanus bacillus a crystallizable toxic sub- stance, called by him tetanin, which was found to kill small animals in very minute doses and w^itli the charac- teristic symptoms of tetanus. More recentl}^ Kitasato and Weyl have obtained the same siibstance, by follow- ing Brieger's method, from a pure culture of this bacil- lus. From a bouillon made from \\ kilogr. of lean beef, with the addition of 25 gm. of i^eptone, they obtained 254 IMMUNITY AND SERUM-THERAPY. 1.7118 g-m. of hydrochlorate of tetaniii. This proved fatal to white mice in six hours in the dose of 0.05 gm., and a dose of 0.105 gm. caused characteristic tetanic con- vulsions and death within an hour. The bacteriolog-ists last named also obtained from their cultures the tetano- toxin of Brieger. Two mice were inoculated subcutane- ously with 0.003 gm. of this substance ; one died at the end of five hours "wdthout the development of tetanic symptoms ; the other survived. In addition to these substances, indol, phenol, and butyric acid were demon- strated to be present in cultures of the tetanus bacillus. The more recent researches of Brieger and Frankel, and of Kitasato, show that the toxic jitomaine discovered by Brieger in 1886 is not the substance to which cultures of the tetanus bacillus owe their great and peculiar pa- thogenic power. The distinguished German chemist and his associate have succeeded in isolating from tetanus cultures a toxalhumhi which is far more deadly than tetanin. Brieger and Colin in more recent investigations (1893) relating to the toxic products of the tetanus bacillus have arrived at the following results : The cultures were made in veal bouillon containing one per cent of peptone and one-fifth per cent, of chloride of sodium. Large quanti- ties of the cultures in this medium were filtered through porcelain filters. The active substance was precipitated from the filtrate by means of a saturated solution of am- monium sulphate. By adding this salt in excess the precipitate is made to rise to the surface and is skimmed off with a platinum spatula. The liquid is removed by placing this upon porous porcelain plates and the crude toxin is dried in a vacuum. It still contains 6.5 per cent, of ammonium sulphate. The tetanus bouillon after filtra- TETANUS. 255 tion is said to be fatal to mice in the dose of 0.00005 c.c. A litre of this bouillon gave about one gramme of the dried precipitate, which produced characteristic tetanic symptoms and death when injected into mice in the dose of 0.0000001 gm. Kitasato in his experiments had previously obtained a tetanus bouillon which was five times as toxic as that used by Brieger and Cohn in their experiments, and which killed mice in the dose of 0.00001 c.c. The dried precipitate obtained by Brieg-er and Cohn con- tained various impurities, including- a certain amount of ammonium sulphate, but was found to kill susceptible animals in the proi)ortion of 0.0000066 g-m. per kilo^m. of body-weight. It was purified without loss of toxic power by placing it in a dialyzer in running water for from twenty-four to forty-eight hours, after which it was dried in vacuo at 20° to 22° C. The purified toxin as thus obtained had a slightly yellowish color, and was in the form of transpar- ent scales, which were odorless, tasted like gum acacia, and were easily soluble in water. The chemical reactions of this purified toxin, according to Brieger and Cohn, show that it is not a true albuminous body. When in- jected beneath the skin of a mouse weighing 15 gm., in the dose of 0.00000005 gm., it causes its death, and one- fifth of this amount gave rise to tetanic symptoms from which the animal recovered after a time. The lethal dose for a man weighing 70 kilogm. is estimated by the authors named to be 0.00023 gm. (0.23 milligm.). Comparing this with the most deadly vegetable alkaloids known it is nearly six hundred times as potent as atropin and one hundred and fifty times as potent as strychnin. Fermi and Pernossi (1894), as a result of an elaborate research, have determined many of the chemical charac- 256 IMMUNITY AND SERUM-THERAPY. ters of tlie tetanus toxin. When in solution it is de- stroyed by a comparatively low temperature (55° C. for one hour) and by exposure to direct sunlight, but the dry powder resists a temperature of 120° C. It has not the properties of an alkaloid, as it is not dissolved by any of the usual solvents of these bodies — the only solvent thus far discovered is said to be water. It resembles the albumins and peptones in its failure to pass throug-h a dialyzing membrane. The authors last referred to con- clude their summary of results as follows : "-The appended table shows that the tetanus poison, like that of diphtheria, in its behavior as regards the action of light, heat, chemical ag-ents, and dialysis, as also its solvents, the ag^ents which precipitate it, and its action upon living- animals, closely resembles the poisons of ser- pents (Naja tripudians, Crotalus, etc.). As to the chemi- cal nature of this group of substances we can at present only say that they rather have the characters of colloidal substances than otherwise, and more nearly resemble the albuminoid bodies than the bases. • We do not, however, reject the verj'^ probable hypothesis that these toxins are acids or bases, or other very unstable, peculiar substances which are closely united with colloidal substances, as is the case, for example, with the alkali and acid albumins and so many other albuminous bodies." While the exact nature of the toxic substance contained in tetanus cultures has not been determined we probably' cannot, at present, do better than to continue to speak of it as a " toxalbumin." Kitasato (1891) was not able to produce immunity in mice by inoculations with minute doses of the poison, or with a filtrate which had been exposed to various degrees of temperature by which its activity was diminished or TETANUS. 2r>7 destroyed. But immunity lasting- for about two months was produced in rabbits by inoculating tliem with the filtrate from a culture of the tetanus bacillus, and subse- quently, in the same locality, with 3 c.c. of a one jjer cent, solution of terchloride of iodine ; this last solution Avas injected subcutaneously in the same dose at. intervals of twenty-four hours for five days. Of fifteen rabbits treated in this way six proved to be immune against large doses of a virulent culture of the tetanus bacillus. The same treatment was not successful in producing immunity in mice or guinea-pigs, but the important discovery was made that a small quantity of blood (0.2 c.c.) from an im- mune rabbit, when injected into the abdominal cavity of a mouse, gave it immunity from the effects of inoculations with the tetanus bacillus. Moreover, mice which were first inoculated with a virulent culture of the bacillus, and, after tetanic symptoms had appeared, received in the cavity of the abdomen an injection of blood-serum from an immune mouse, were preserved from death. The power of the blood of an immune animal to neutralize the tetanus poison was further shown b^^ mixing the fil- trate from a virulent culture with blood-serum from an immune animal and allowing it to stand for twenty-four hours ; a dose three hundred times greater than would have sufficed to kill a mouse proved to be without effect after such admixture with blood-serum ; as before stated, the blood-serum of animals Avhicli are not immune has no effect upon the poison. The duration of immunity induced in this way was from forty to fifty days. Blood-serum from an immune rabbit, preserved in a cool, dark room, retains its power of neutralizing the tetanus poison for about a week, after which time it gradually loses it. Having found that chickens have a natural immunity 17 258 IMMUNITY AND SERUM-THERAPY. against tetanus, Kitasato made experiments to ascertain whether their blood-serum would also neutralize the tetanus poison ; the result was neg-ative. That the tetanus poison is present in the blood of in- dividuals who die from tetanus has been jjroved by Kitasato by .injecting a small quantity (0.2 to 0.3 c.c.) of blood from the heart of a fresh cadaver into mice ; the animals develop typical tetanic symptoms and die in from twenty hours to three days. Tizzoni and Cattani have (1891) reported results simi- lar to those obtained by Kitasato. By reiieated inocula- tions with gradually increasing doses of the tetanus poison they succeeded in making a dog and two pigeons immune, and found that blood-serum from this immune dog, in very small amount, completely destroyed the toxic power of a filtrate from cultures of the tetanus bacillus — one to two drops of serum neutralized 0.5 c.c. of filtrate after fifteen to twenty minutes' contact. They also ascertained that small amounts of blood-serum from this immune dog injected into other dogs or white mice j)rodiiced immunity in these animals ; but the}' were not able to produce immunity in guinea-pigs or rabbits by the same method. In a later communication (May, 1891) Tizzoni and Cat- tani give an account of their experiments made with a view to determining the nature of the substance in the blood-serum of an immune animal which has the power of destroying the toxalbumin of tetanus — " tetanus anti- toxin." They found, in the first place, that this antitoxin in blood-senim is destroyed in half an hour by a tem- perature of 68° C ; further, that it does not pass through a dialyzing membrane ; that it is destroyed by acids and alkalies. As a result of their researches thev conclude TETANUS. 259 that it is an albuminous substance having the nature of an enzyme. Vaillard has succeeded in producing immunity in rab- bits by repeated injections into the circulation of filtered cultures — in all 20 c.c. — which had been exposed for one hour to a temperature of 60° C. At a temperature of 65° C. both the toxic and the immunizing- action is de- stroyed. Behring (1892) gives the following account of a method which he has successfully employed for producing im- munity in large animals — especially in horses : A culture of the tetanus bacillus is made, in bouillon, of such toxic potency that 0.75 c.c. will kill a rabbit in three or four days. To 200 c.c. of this culture he adds carbolic acid in the proportion of 0.5 per cent, for the purpose of preserv- ing it. The horse first receives a subcutaneous injection of 10 c.c. of this culture fluid to which terchloride of io- dine (I CI3) has been added in the proportion of 0.25 per cent. ; at the end of eight days 20 c.c. of the same mixt- ure are given ; again in eight days the dose is repeated ; then, after an interval of three days, 30 c.c. of the same mixture. Following this, at an interval of eight days, he gives two injections of 30 c.c. each of a mixture contain- ing one-half the quantity of I CI, (0.175 per cent.). The proportion of the iodine terchloride is then reduced to 0.125 per cent., and two doses of 20 c.c. each are given. Finally the culture-fluid is administered in the dose of 0.5 c.c, and this dose is doubled every five days. Before giving the first dose of culture-fluid without the addition of I CI3, the immunizing value of the blood-serum of the horse is tested upon mice, and if it falls below 1 to 100 a dose of 0.25 c.c. is given instead of the larger dose (0.5 c.c.) above mentioned. 260 IMMUNITY AND SERUM-THERAPY. Schiitz (1892) lias applied Beliring's method to a con- siderable number of horses and sheep, and arrives at the conclusion that it is a reliable method of protecting these animals against infection with living tetanus bacilli and against the toxic action of filtered cultures ; that the de- gree of immunity and the antitoxic j)ower of the blood- serum increases as larger doses are gradually given. Ac- cording to Behring the immunizing value of blood-serum from a horse treated in this way is very high. As tested on mice it may be 1 to 200,000, or even more. According to his calculations a serum having a value of 1 to 100,000, as tested on mice, should be given to a man weighing 50 kilogr. in the quantity of 50 c.c, given in the course of two days, in order to insure immunity. The same author in a subsequent paper (1892) gives details as to the method of estimating the therapeutic value of serum from an immune animal. He first calls attention to the fact that the only reagent by which the antitoxic potency of this serum can be tested is the body of a living animal. The test animal selected is the white mouse. When the statement is made that a serum has the value of 1 to 1,000,000, he means that by an experi- mental test, made upon white mice, it has been ascer- tained that these animals are protected from fatal infec- tion with the minimal lethal dose of a tetanus culture by the use of 0.00002 gm. of the serum for a mouse weighing 20 gm. For the cure of tetanus in the mouse, after the first symptoms of the disease have appeared, a dose at least one thousand times as great as the immunizing dose is required, and the more advanced the progress of the case the greater the dose must be. A serum of the strength above indicated, if used for the treatment of a case of tetanus in man, should, according to Behring, be TETANUS. 261 employed in doses amounting' altogether to at least 100 c.c. — g-iven inside of twenty-four hours in doses of 20 c.c. each. For persons sixteen years old he would give doses of 10 c.c, and for children under six, 5 c.c. at a dose. The serum of this strength which he had prepared for testing- its curative value on man was preserved by the addition of 0.5 per cent, of carbolic acid. Rotter (1892) reports a case successfully treated by Behring's serum. In all 250 c.c. was administered sub- cutaneously. The case was not, however, one of the most severe forms of the disease. Brieg-er and Ehrlich (1892) have succeeded in immuniz- ing' g-oats by means of gradually increasing- doses of a culture of the tetanus bacillus in thymus bouillon. The amount g-iveu at first was 0.2 c.c, and this was gradually increased to 10 c.c. At the end of thirty-seven days the animal was found to be immune against virulent cultures, and the important fact was demonstrated that the im- munizing substance (antitoxin) was present in its milk. A mouse which received 0.1 c.c. of the milk of this goat in the peritoneal cavity proved to be immune ag-ainst infection as a result of inoculation with a tetanus culture. The immunizing- value of the milk from this goat was found to be 1,600. That is, a dose of 0.2 c.c, which was equal to 1 to 109 of the body-weig-ht of the animal, protected a mouse from sixteen times the fatal dose of a tetanus culture. After precipitation of the casein the milk still preserved its antitoxic power unim- paired, and by concentrating- it in vacuo a fluid was ob- tained which proved to have an immunizing- value of 5,000. • In a later communication (1893) Brieger and Colin give the results of additional experiments with the milk 262 IMMUNIXr AND SERUM-THERAPY. of immunized g-oats. Animals were chosen which were two or three years old and had given birth to young- a few weeks before the inoculations were commenced. It having been previously shown by Ehrlich that the pre- cipitated tetanus toxin from cultures could be success- fully used to immunize guinea-pigs, the same substance was employed in these experiments. The treatment was commenced with a dose of 0.00001 gm., which was carefully increased to 0.00007 gr., the injections being made at intervals of four days. But this proved to be too much, and the animal died of typical tetanus after the last dose. In a subsequent experiment Brieger and Cohn succeeded in immunizing a goat in a month and a half so that the animal finally withstood a dose of 0.06 gm., but this animal ceased to give milk, became anae- mic, and finally died. The authors therefore resorted to a different method which had previously been successfully employed by Ehrlich, Behring, and others. Cultures of the tetanus ba- cillus in bouillon were heated to 65° C. for half an hour, and then used for immunizing two goats. After five weeks' treatment the animals resisted doses of the pre- cipitated toxin, which were gradually increased to 10 gm., at which time the treatment had been carried on for nearly six months and the antitoxic value of the milk was found to be 90,000 immunization units. The method of determining antitoxic values adopted by Brieger and Cohn is the following : They had found by carefully conducted experiments that their precipi- tated toxin {Rohgifte) killed a mouse weighing 20 gm. in the dose of 0.0000003 gm.,but failed to kill when injected in the dose of 0.0000002 gm. The first-mentioned dose was therefore accepted as the minimum fatal dose for an TETANUS. " 263 animal weig-liing- 18 to 20 g-m., and the object in view was to find the minimum amount of milk required to prevent the toxic action of such a dose. The antitoxin was obtained from the g-oat's milk by precipitation with ammonium sulphate, thirty-two per cent. ; the precipitate was again dissolved and treated with a solution of basic acetate of lead ; this salt does not precipitate the antitoxin when the solution is slight- ly alkaline ; the voluminous precipitate produced by the lead acetate is filtered out and repeatedly washed with water ; the filtered fluid and wash-water are again treated with ammonium sulphate, added to saturation, and the resulting precipitate is dissolved in a small quantity of water ; a precipitate is again obtained by saturation with ammonium sulphate, and this is dried upon porcelain plates in a vacuum. The ammonium sulphate remaining could not be removed by dialysis, as experiment showed that a considerable loss of the antitoxin occurred in a di- al.yzer placed in running water. But by shaking- up the dry powder in chloroform the heavy salt sank to the bot- tom and the purified antitoxin floated on the surface and could be recovered by skimming it off. The powder thus obtained consisted of a mixture of various substances, in- cluding the antitoxin, and wdien obtained from milk hav- ing an antitoxic value of 90,000 it was found to have a value of 25,000,000 immunization units. By further puri- fication a still higher value was obtained (55,000,000). In experiments on mice a dose ten thousand times as great as was necessary to produce immunity proved to exercise a curative power— i.e., a dose of 0.02 gm. for a mouse weighing 20 gm. saved it from being killed by double the minimum fatal dose of the tetanus toxin, after tetanic symptoms had been developed. 264 IMMUNITY AND SERUM-THERAPY. Reference has been made to the production of immu- nity by the use of cultures made in thymus bouillon. This was made known through the experiments of Brie- ger, Kitasato, and Wassermann (1892). The thymus bouillon is made from the thymus glands of calves, which are chopped fine in a hash-machine and covered with an equal volume of distilled water. The mixture is stirred for some time and then placed in an ice-chest for twelve hours ; the liquid is then obtained by filtration through gauze with pressure — by means of a flesh-press machine. A turbid, slimy fluid is thus obtained, which is diluted with an equal volume of water and made slightly alkaline by the addition of soda solution. It is then sterilized at 100° C. for fifteen minutes. As a result of this the liquid has a grayish-brown color, and some large flocculi in sus- pension, which are removed by passing it through fine linen. The fluid is then of a milky opalescence. It is next placed in test tubes and again sterilized. The tet- anus bacillus when cultivated in this medium does not form spores, and the toxic potency of the culture is very much reduced — 1 to 5,000 to 1 to 3,000 of the toxic potency manifested by cultures of the same bacillus in ordinary media. Inoculations with cultures in thymus bouillon were found to kill mice in the dose of 0.5 c.c, while small- er amounts failed to kill and caused the animals to be immune. A culture in ordinary bouillon was fatal to mice in the dose of 0.001 c.c. Experiments on rabbits (thirty-five) gave a uniformly successful result in immunizing these animals. Immu- nity was established in the course of two weeks, and the blood-serum of these animals tested on mice showed an antitoxic value of 1,000. Reference has already been made to the earlier re- TETANUS. 265 searches of the Italian investigators, Tizzoni and Cat- tani. These have been followed by additional investi- gations, the results of virhich have been reijorted in numerous published iiapers. The authors named have ascertained that when kept in a cool place (15° to 25° C.) the blood-serum of immune rabbits retains its antitoxic power for several months, and the antitoxin, obtained by j)recipitation with alcohol, kept in a dry condition for more than ten months, was found to preserve its original activity. Having succeeded in their earlier experiments in immu- nizing rabbits and dogs, Tizzoni and Cattani (in 1893) proceeded to experiment upon horses, and were equally successful with these animals. As a result of numerous injections with an attenuated virus, continued for a pe- riod of ninety-seven days, they established an immunity which was tested by inoculating the animal with 10 c.c, of a gelatine culture, of which l-200th part of a drop killed a white mouse. The antitoxic value of the blood- serum of this horse was 1 to 5,000,000— he., 1 gm. of this serum would immunize 5,000,000 gm. of mice, or 250,000 mice weighing 20 gm. each. In a later communication (1894) the authors named report that after freely bleed- ing immunized horses, and allowing them to rest for one or two months, and then again treating them with small doses of tetanus cultures, the blood-serum soon becomes as active as before the bleeding. The greatest antitoxic power was manifested from twenty to twenty-three days after the completion of the protective inoculations, and a serum was obtained possessing a value of 1 to 10,000,000. According to the authors named the precipitated (by alcohol) and purified antitoxin from such a serum, judg- ing from their experiments on lower animals, should cure a case of tetanus in man in the dose of 40 to 50 ctgr. 266 IMMUNITY AND SERUM-THERAPY. The authors last nientioneLl have reported (1892) thut the young- of immuue i:)arents have a certain degree of m- herited immunity. And the more recent experiments of Ehrlich and Hiibener have confirmed this so far as the inheritance of immunity from the mother (in mice) is concerned ; but their results did not show any immunity in the young- when only the father had been rendered immune ; and the immunity inherited from the mother only lasted for two or three months after birth. Serum-therapy in Man. The evidence has already been presented in support of the therapeutic value of the blood-serum from animals immunized against tetanus, and of the precipitated anti-^ toxin from such serum. It only remains to present the results of the same treatment as applied to tetanus in man. Dr. Rudolph Schwarz, Assistant to the Surgical Clinic of Padua, reported the first successful case, treated with an antitoxin prepared by Professor Tizzoni from the blood-serum of a dog which he had immunized against tetanus. The following is an abstract of the case as re- ported by Dr. Schwarz : " The patient was a peasant boy, aged fifteen years, who wounded himself in the left forearm while attempt- ing to cut a walnut which he had picked up from the ground. There was considerable hemorrhage, which was controlled by the application of a wad of spider's web and a bandage. A return of the hemorrhage induced the parents to take the boy to the Surgical Clinic at Padua. There, under appropriate treatment, the hemorrhage was arrested and the wound healed. Two weeks after receiv- ing the wound symptoms of tetanus were developed. The patient was admitted to hospital three days later, at TETANUS. 267 which time the jaws were immovable and the muscles of the arm in a state of tetanic rigidity, while other muscles of the trunk and extremities were also involved. Treat- ment with chloral and warm baths was without effect in arresting- the progress of the malady, and on the 16th of September, nine days after admission, the patient had eight or nine tetanic convulsions during the night, ac- companied by difficult respiration and slight opisthot- onos. No improvement having occurred under the treatment employed, it was decided to test the value of tetanus antitoxin, which had been sent for the purpose by Professor Tizzoni, who had obtained it from the blood-serum of a dog which had been rendered ' strongly immune ' against tetanus. On the afternoon of Septem- ber 18th, 15 ctgr. of this antitoxin, dissolved in water, were injected beneath the skin. The same quantitj^ was injected the following day. On the morning of the 20th the patient Avas decidedly better ; on the afternoon of this day he received 25 ctgr. of the antitoxin, and the same amount on the following day. After each injec- tion there was a notable fall in the temperature. The patient continued to improve, and on the 23d all symp- toms of tetanus had disappeared." In a postscript to his communication Dr. Schwarz states that he has private information of two other pa- tients who have recovered from tetanus under the same treatment — one in the hospital at Colle di Val d'Elsa (Tuscany), treated by Dr. Pacini ; the other in the Surgi- cal Clinic at Innsbruch, treated by Professor Nicoladoni. Eenon (1892) reports two cases treated by subcutaneous injections of the blood of immune rabbits (one received in all 57 c.c, the other 80 c.c). Both resulted fatally, but an amelioration of the symptoms was noted in both cases after each injection of the antitoxic serum. Baginsky (1892) reports the case of a child, nine days 268 IMMUNITY AND SERUM-THERAPY. old, in which tetanus was developed as a result of infec- tion through the navel. The case was treated by Kita- sato with serum from an immune rabbit in doses of 0.1 to 0.4 C.C., repeated daily (total amount used 1.5 c.c). The child died on the thirteenth day, and Baginsky infers that the doses employed were too small. Additional cases have been reported as successfully treated by several Italian physicians (Casali, Finotti, Gagliardi, Pacini, Taruffi). In all of these cases the dry antitoxin supplied by Tizzoni and Cattani, and obtained by them from the serum of immunized dogs, was em- ployed. The dry powder in the dose of 0.25 gm. was dissolved in water and injected subcutaneously. In an- other case reported by Tizzoni 40 c.c. of rabbit serum was used. Recovery occurred in all of the cases. In one very severe case (the tenth treated) reported by Finotti, improvement occurred after the administration of the antitoxin, but the supply was exhausted and the tetanic symiDtoms again became more severe ; upon the receipt of an additional supply and a renewal of the treatment improvement again occurred and the case recovered. The eleventh successful case is reported by Dr. Gattai, of Pisa, the twelfth (1893) by Lesi, of Imola. BIBLIOGRAPHY. Behring, E. : Das Tetanusheilserum uud seine Anwendung auf tetanus- kranke Meuscben. Leipzig, 1892 (Thieme). Ueber Iramunisirung uud Heilung von Versuclisthieren bcim Tetanus. Zeilscbr. fiir Hyg., XII., 45, 1893. Bebring und Knorr : Ueber den Immunisirungswertb und Heilwert des Tetanusbeilserums bei weissen Mausen. Zeitscbr. fiir Hyg., XIII., 407, 1893. Behring und Kitasato : Ueber das Zustandekommen der Dipbtherie- Immunitat und der Tetanus-Immunitat. Deutsche med. Wchschr., 1890, No. 49. TETANUS. 269 Briegei-und Colin : Untersuchungen tiber das Tetanusgift. Zeitschr. ftlr Hyg., XV., 1, 1898. Beitrage zur Konzeiitrierung gegen WunSstarrkriimpjI scliiizen- den substauz aus der jVIilch. Zeitschr. f. Hyg., 1893, 489. Courniont, J., et Doyon, M. : La substance toxique qui engendre le te- tanos resulte de Faction sur I'orgauisme recepteur d'un ferment soluble fabrique par le bacille de Nicolaiier. Compte-rendu, 8oc. de Biol., 1893, p. 394. De la production du tetanos chez la poule, et de la creation arti- ficielle de rimraunite chez cet animal. Compte-rendu, Soc. de Biol., 1893, p. 841. Ehrlich, P., und Htibeuer, W.: Ueber die Vererbnng der Imniuiiitat bei Tetanus. Zeitschr. f. Hyg., XVIH., 51, 1894. Fedoroff, S. : Wirkt das Tetanusaiititoxin auch giftzerstorend. Cen- tralbl. f. Bacteriol., XVI., 484, 1894. Fermi, C, und Pernossi, L. : Ueber das Tetanusgift. Centralhl. f. Bak- teriol., XV., 303, 1894. Ueber das Tetanusgift. Zeitschr. ftir Hyg., XVI., p. 386, 1894. Kitasato : Experimentelle Untersuchungen iiber das Tetanusgift. Zeitschr. fiir Hygiene, Bd. X., 1891, p. 267. Heilversuche an Tetanuskranken Thieren. Zeitschr. fflrHyg., XII., 256, 1893. Klipstein, E. : Ueber die Wirkung giftfreierTetanuskulturen. H3'gien. Rundschau, 1898, p. 1. Renou : Deux cas de tetanos traites par des injections de sang antitox- ique. Ann. de I'Inst. Pasteur, 1893, p. 383. Schiitz : Versuche zur Immunisirung von Pferden und Schafen gegen Tetanus. Zeitschr. fiir Hyg., XII., 58, 1893. Tizzoni e Cattani : Ueber das Tetanusgift. Centralbl. ftlr Bakteriol. , Bd. VIII. , 1890, p. 69. Ueber die Art einen Thiere die Iramunitat gegen Tetanus zu tlbertragen. Centralbl. fiir Bakteriol., Bd. IX., 1891, p. 189. Ueber die Eigenschaften des Tetanus-Antitoxins. Centralbl. ftlr Bakteriol., Bd. IX., 1891, p. 685. Fernere Untersuchungen tiber das Antitoxin des Tetanus. Cen- tralbl. fUr Bakteriol., Bd. X., 1891, p. 33. Alcune questioni relative all' immunlta pel tetano. Riforma Med., 1893, No. 193,. 193. Sulla transmissione ereditaria dell' immunita contro il tetano. Riforma Med., 1893, No. 94. Suir importanza della milza nell' immunizzazione sperimentale del coniglia contro il tetano. Riforma Med., 1893, No. 49. Esperienze sulla vaccinazioue del cavallo contro 11 tetano. La Rif. Med., 1893, No. 131. 270 IMMUNITY AND SERU3I-TIIERAPY. Tizzoni e Cattani : Weitere Experiraentelle Untersucliungen tiber die Imniunitat gegeu Tetanus. Berl. klin. AVocbenschr., 1893, p. 1185, 1215, 1245, 1265. Neue Unteisucbungen ul)er die Vaccination des Pferdes gegen Tetanus. Berl. klin. Wcbscbr., 1894, No. 32. Nuove esperienze sulla vaccinazione del cavallo contro il te- tano. Gazz. d. Ospit., 1894, p. 505. Weitere Experimentelle Untersucbungen tiber die Inimunitiit gegen Tetanus. Berl. klin. Wcliscbr., 1894, p. 64. Trevisan, A. : Sulla inalterata virulenza del materiale tetanigeno con- servato in glicerina. Rev. Veneta di Sci. Med., 1892, p. 129. Vaillard : Sur rimmuuite contre le tetanos. Corapte-rendu, Sec. de Biol., 1891, No. 7. Sur les proprietes du serum des aniraaux refractaires an teta- nos. La Semaine Med., 1891, p. 254. Sur rinoculation aux animaux du bacille tetanique depourvu de toxine. Le Bull. Med., 1891, p. 901. Sur quelques points concernant rimmunite contre le tetanos. Ann. de I'lnst. Pasteur, 1892, p. 676. De Paction des bumeurs d'un animal immunise contre le tetanos sur le virus de cette maladie. Ann. de I'lnst. Pasteur, 1892, p. 676. Wernicke : Ueber Bebring's Blutserumtherapie bei Tetanus. Abst. in Centralbl. Mr Bakteriol., XV., 898, 1894. Wladimiroflf, A. : Ueber die Antitoxinerzeugende und Immunisirende Wirkung des Tetanusgiftes bei Tbieren. Zeitscbr. ftLr Hyg., XV., 405, 1893. XX. TUBERCULOSIS. The announcement of the discovery of the tubercle bacillus was made by Koch, in March, 1882, at a meeting" of the Physiological Society of Berlin. At the same time satisfactory experimental evidence was presented as to its etiological relation to tuberculosis in man and in the susceptible lower animals, and its principal biologi- cal characters were given. This achievement, the result of patient and intelligent scientific investigation, will always rank as one of the most important in the history of medicine. The previ- ous demonstration by Villemin (1865) — confirmed by Cohnheim (1877) and others — that tuberculosis might be induced in healthy animals by inoculations of tubercu- lous material, had paved the way for this great discov- ery, and advanced pathologists were quite prepared to accept it. The more conservative have since been obliged to yield to the experimental evidence, which has received confirmation in all parts of the world. To-day it is generally recognized that tuberculosis is a specific infectious disease due to the tubercle bacillus. As evidence of the thorough nature of Koch's personal researches in advance of his first public announcement, we give the following resume of his investigations : In nineteen cases of miliary tuberculosis the bacilli 272 IMMUNITY AND SERUM-TIIEKAPY. were found in the tubercular nodules in every instance ; also in twenty-nine cases of pulmonary phthisis, in the sputum, in fresh cheesy masses, and in the interior of recently formed cavities ; in tuberculous ulcers of the tongue, tuberculosis of the uterus, testicles, etc. ; in twenty-one cases of tuberculous — scrofulous — lymi^hatic glands ; in thirteen cases of tuberculous joints ; in ten cases of tubercular bone affections ; in four cases of lupus ; in seventeen cases of jyerhucht in cattle. His ex- perimental inoculations were made upon two hundred and seventy-three guinea-pigs, one hundred and live rab- bits, forty-four field-mice, twenty-eight white mice, nine- teen rats, thirteen cats, and upon dogs, pigeons, chick- ens, etc. Very extensive comparative researches were also made, which convinced him that the bacillus which he had been able to demonstrate in tuberculous sputum and tissues by a special mode of staining was not to be found in the sputa of healthy persons, or of those suffer- ing from non-tubercular pulmonary affections, or in or- gans and tissues involved in morbid processes of a differ- ent nature. The tubercle bacillus is a stncf j)arasite, and its bio- logical characters are such that it could scarcely find natural conditions, outside of the bodies of living ani- mals, favorable for its multiplication. It therefore does not grow as a saprophyte under ordinary circumstances. But it has been noted by Eoux and Nocard that when it has been cultivated for a time in artificial media con- taining glycerin it may grow in a plain bouillon of veal or chicken, in which media it fails to develop when in- troduced directly from a culture originating from the body of an infected animal. This would indicate the possibility of its acquiring the ability to grow as a sapro- TUBERCULOSIS. 273 pliyte ; and we can scarcely doubt that at some time in the past it was a true saprophyte. The experiments of Nuttall indicate that the bacillus may multiply, under favorable temperature conditions, in tuberculous sputum outside of the body. And it is extremely probable that multiplication occurs in the muco-purulent secretion which accumulates in pulmonary cavities in phthisical patients. In these cavities its development may, in a certain sense, be reg-arded as saprophytic, as it feeds upon non-living* org-anic material. Metschnikoff states that when kept at a temperature of 42° C. for some time the tubercle bacillus underg-oes a notable diminution in its i3athog"enic power, and that when kept at a temperature of 43° to 44° C. it after a time only induces a local abscess when injected subcu- taneously into gruinea-pigs. The experiments of Lote also indicate that an " attenuation of virulence " has oc- curred in the cultures preserved in Koch's laboratory, orig'inating" in 1882 from the lungs of a tuberculous ape. The author named made experiments with cultures from this source (ninetieth to ninety-fifth successive culture), and at the same time with a culture obtained from Koux, of Pasteur's laboratory. Rabbits inoculated with cultures from the last-mentioned source developed a hectic fever at the end of two weeks, and died tuberculous at the end of twenty-one to thirty-nine days. Twelve rabbits were inoculated with the cultures from Koch's laboratory ; the injections were made either subcutaneously, into a vein, into the pleural cavity, or into the cavity of the abdo- men. No elevation of temperature occurred in any of the animals, and they were found at the end of a month to have increased in weig-ht. At the end of six weeks one of them was killed and tubercular nodules were 18 274 IMMUNITY AND SERUM-THERAPY. found in various organs. The remaining animals were killed at the end of one hundred and forty-four to one hundred and forty-eight days. The two inoculated sub- cutaneouslj^ presented no sign of general tuberculosis, but a small yellow nodule containing bacilli was found at the point of inoculation. Those inoculated by injec- tion into a vein showed one or two nodules in the lungs containing a few bacilli. In Koch's original experiments rabbits were killed by intravenous inoculation of his cult- ures in from thirteen to thirty-one days. That this at- tenuation of virulence depends upon a diminished pro- duction of a toxic product to which the bacillus owes its pathogenic power appears to be very certain, in view of the fact that the late cultures in a series have a more vigorous and abundant development than the more patho- genic cultures obtained directly from the animal body. The discovery by Koch of a toxin in cultures of this ba- cillus, which is soluble in glycerin, and which in very minute doses produces febrile reaction and other decided symptoms when injected subcutaneously into tubercu- lous animals, must rank as one of the first importance in scientific medicine, whatever the final verdict may be as to its therapeutic value in tubercular diseases in man. The toxic substance contained in Koch's glycerin ex- tract from cultures of the tubercle bacillus, now gener- ally known under the name of tuberculin, is sohible in water, insoluble in alcohol, and passes readily through dialyzing membranes. It is not destroyed by the boil- ing temperature. According to the chemical examina- tion of JoUes, the " lymph " contains fifty per cent, of water and does not contain alkaloids or cyanogen com- pounds. It contains albuminates, which are thrown down as a voluminous white precipitate by tannic acid, TUBERCULOSIS. 275 and are redissolved by hot water containing sodium chloride and very diluted potash solution. The elemen- tary analysis gave N 5.90 per cent., C 35.19 per cent., and H 7.02 per cent. The results obtained are believed to show that the active substance present in the lymph is a toxalbumin. In experiments made with Koch's lymph in Pasteur's laboratory by Bardach, a very decided ele- vation of temperature was produced in tuberculous guinea-pigs by the subcutaneous injection of 0.1 gm., and a fatal result by the injection of 0.2 to 0.5 gm. In man a decided febrile reaction is produced in tuberculous pa- tients by very much smaller doses — 0.001 c.c. Hammerschlag, in his chemical researches, found that the tubercle bacillus yields a larger proportion of sub- stances soluble in alcohol and ether than any other ba- cilli tested (twenty-seven per cent.). The alcoholic ex- tract contains fat, lecithin, and a toxic substance which produces convulsions in rabbits and guinea-pigs. The portion insoluble in alcohol and ether contains cellulose and an albuminoid substance. No ptomaines were found, but a toxalbumin was isolated, which caused an elevation of temperature in rabbits of 1° to 2° C, lasting for a day or two. Koch (1891) has given a full account of his method of preparing crude tuberculin, and also the process by which he obtains from this a tuberculin which appears to be pure, or nearly so. To obtain considerable quantities of the crude product the tubercle bacillus is cultivated in an infusion of calves' flesh, or of beef extract to which one per cent, of peptone and four to live per cent, of glycerin have been added. This culture liquid must be made slightly alkaline, and it is placed in flasks with a flat bottom, which should not be more than half filled— 27G IMMUNITY AND SERUM-TIIEIIAPY. 30 to 50 c.c. The inoculation is made upon the sur- face with small masses from a culture upon blood-serum or g-lycerin agar. By accident Koch discovered that these masses fioating upon the surface give rise to an abundant development, and to the formation of a toler- ably thick and dry white layer, which finally covers the entire surface. At the end of six to eight weeks develop- ment ceases, and the layer after a time sinks to the bot- tom, breaking up meanwhile into fragments. These cultures, after their purity has been tested by a micro- scopical examination, are poured into a suitable vessel and evaporated to one-tenth the original volume over a water-bath. The liquid is then filtered through porce- lain. The crude tuberculin obtained by this process con- tains from forty to fifty per cent, of glycerin, and conse- quently is not a suitable medium for the develojDment of saprophytic bacteria, if they should by accident be intro- duced into it. It keeps well and preserves its activity indefinitely. From this crude tuberculin Koch has obtained a white precipitate, with sixty per cent, alcohol, which has the active properties of the crude tuberculin as originally prepared. This is fatal to tuberculous guinea-pigs in doses of 2 to 10 milligm. It is soluble in water and in glycerin, and has the chemical reactions of an albumi- nous body. In preparing it one volume and a half of absolute alcohol is added to one volume of the crude tuberculin, and, after stirring it to secure uniform admixt- ure, this is put aside for twenty -four hours. At the end of this time a flocculent deposit will be seen at the bot- tom of the vessel. The fluid above this is carefully poured off ; and an equal quantity of sixty per cent, alco- hol is poured into the vessel for the purpose of washing TUBEKCULOSIS. 277 the precipitate. This is again allowed to settle, and the procedure is repeated three or four times, after which the I)recipitate is washed with absolute alcohol. It is then placed upon a filter and dried in a vacuum exsiccator. The " tuberculocidin " of Klebs is a purified tuberculin obtained by precipitation with alcohol. The precipitate is washed in chloroform and then dissolved in a mixture of carbolic acid and glycerin. Bujwid (1894) prepares tuberculin as follows : He uses cultures on glycerin agar or in glycerin bouillon which have been kept at a suitable temperature for five to eight weeks. The glycerin-agar cultures are treated with dis- tilled water by which the tuberculin is extracted. After adding the water the test-tubes are kept in a cool place for twenty-four hours, and this is repeated two or three times. The extract from the agar cultures or the bouil- lon cultures is then sterilized by exposure for from five to ten minutes to a temj)erature of 100° C ; then filtered through a Chamberland filter ; then evaporated at a low temperature to a sirup -like consistence. When this crude tuberculin is dropped into ten times its volume of strong alcohol a brown precipitate is thrown down which contains the active principle. From the tubercle bacilli obtained by filtering his cultures Bujwid also obtained an active substance which in the dose of 2 milligm. caused an elevation of 2° C. in the temperature of an infected guinea- pig. This substance was obtained by digesting the ba- cilli for two months in glycerin and water (three per cent, of glycerin), filtering and evaporating the extract, and precipitation in six volumes of ninety-five per cent, alcohol. The precipitate when dried was in the form of a white powder. Helman (1894) obtains tuberculin from potato cultures. 278 IMMUNITY AND SERUM-TIIEIIAP V. The sections of potato are neutralized by leaving- them for half an hour in a solution of one-half to one per cent, of bicarbonate of soda, after which they are steril- ized for twenty minutes in the autoclave at 120° C. The best results were obtained when the potatoes were wet with a five per cent, solution of g-lycerin. The sections of potato were iDlaced in Petri's dishes upon blotting- paper wet with a sublimate solution, and the dishes con- taining* the cultures were surrounded with cotton wet with the same solution. The cultures w^ere subsequently treated with distilled water, to extract the active princi- ple, which was also obtained from the bacilli bj^ mixing- them with glycerin in the proportion of 1 to 10. Numerous experiments have been made with dead tu- bercle bacilli, as well as with the toxic products devel- oped in cultures. Hericourt and Richet (1890) found by experiment that old cultures heated to 80° C, several days in succession, when injected into a vein in rabbits, in the dose of 10 to 20 c.c, caused the death of these animals. Smaller doses from which the animals recovered seemed to make them less susceptible to infection than control animals, but the number of experiments was too limited to establish this as a fact. In a subsequent (1891) com- munication the authors named claim to have succeeded in immunizing rabbits by injecting filtered and sterilized cultures of the tubercle bacillus, either subcutaneously (5 to 15 c.c.) or into a vein (20 to 40 drops). The injections were repeated every second or third day for a period of fifteen days, after which the test inoculation was made with a culture, obtained from a tuberculous cow in one series, and from tuberculous fowls in another. Four vac- cinated rabbits in the first series escaped general tubercu- losis, while four out of eight control animals died tuber- TUBERCULOSIS. 279 culous. In the second series five vaccinated animals resisted infection and three out of four control animals died tuberculous. De Schweinitz has recently (1894) reported the results of experiments with attenuated cultures of the tubercle bacillus, and has, apparently, succeeded in conferring im inunity upon guinea-pigs by inoculations with such cult- ures. Klebs (1891), in experiments on guinea-pigs and rab- bits, convinced himself that the fatal result of an inocula- tion with tubercle bacilli (in the cavity of the abdomen or subcutaneously in guinea - pigs, and in the eye in rabbits) was greatly delayed by injections of Koch's tuberculin (0.3 to 0.5 c.c.) either before or after infec- tion. Baumgarten (1891), in experiments upon rabbits inocu- lated with tubercle bacilli in the anterior chamber of the eye, failed to obtain favorable results from treatment with Koch's tuberculin given in considerable doses (0.5 to 1 gm.) either before or after infection. The results reported in the same year by Gramatschi- koff, b}^ Popoff, by Alexander and by Gasparini and Mer- canti, were also unfavorable as regards an immunizing or curative effect from inoculations of tuberculin in rabbits. Diinitz, on the contrary, arrives at the conclusion that when early treatment is instituted iris tuberculosis may be arrested and cured, and the more recent experiments of Trudeau (1893) give support to this conclusion. Baum- garten, however, insists that the tuberculin treatment does not prevent metastasis to the lungs after inoculations in the anterior chamber of the eye. Pfuhl (1891) treated forty-seven infected guinea-pigs and at the date of his report forty -four had died tuber- 280 IMMUNITY AND SERUM-TIIEKAPY. culoiis, but the date of death was somewhat postponed by the treatment. The auimals not treated succumbed at the end of eight weeks (average of all controls), and those treated with small doses of tuberculin lived, on the average, ten weeks. With larger doses still more favor- able results were obtained — four lived on an average twelve weeks, and three were still living, eleven, fifteen, and sixteen weeks after infection, at the date of jjubli- cation. Kitasato (1892) also obtained favorable results in the treatment of infected guinea-pigs, and arrives at the con- clusion that guinea-pigs Avhich have been cured by the treatment are not susceptible to a second infection, for a certain time at least. Bujwid (1892), in experiments upon guinea-pigs, found that infected animals which received from 0.05 to 0.1 gm. of tuberculin within three hours showed an elevation of temperature of 1.5° to 2° C. Thirteen infected guinea- pigs treated with tuberculin lived from two and a half to eight months, while all of the control animals (eighteen) died in from six to nine weeks. The animal which sur vived eight months was found not to be tuberculous but presented evidence of recovery from a former tuberculous process. In two rabbits inoculated in the anterior cham- ber the iris tuberculosis was favorably influenced by the tuberculin treatment, but general infection occurred and the animals died about the same time as the controls. Three apes were treated without any apparent result ; they all died within two months after infection. The experiments of Gramatschikoff, Czaplewski and Roloff, and of Yamagiva, published in 1892, show that the tuberculin treatment does not cure tuberculous infection in inoculated guinea-pigs and rabbits, and that the TUBERCULOSIS. 281 bacilli retain their vitality in such animals in spite of the most persistent treatment. Hericonrt and Ricliet (1892), in experiments made for the purpose of immunizing animals against tuberculous infection, failed to obtain positive results in the most susceptible species — guinea- jiigs, rabbits, and apes — but claim to have succeeded in immunizing dogs by intra- venous injections of cultures of the bacillus of tubercu- losis in fowls. Animals which had been so treated after an interval of two to six mouths received an intravenous injection of 1 c.c. of a culture of the bacillus tuberculosis from man. This was fatal to " non-vaccinated " dogs, as a rule, in about three weeks, but the " vaccinated " animals survived the injection. The results obtained by Trudeau (1893) are of such in- terest that we shall quote in exteiiso what he says with reference to x)reventive inoculations : " Antitubercular inoculation was first tried by Falk in 1883, and all attempts in this direction have resulted un- til recently in but an unbroken record of failures. In 1890 I added my name to the list of those who found it impossible to produce immunity in animals by this method. In 1890, Martin and Grancher, and Courmont and Dor, claimed to have iiroduced in rabbits a certain degree of immunit}^ by previous inoculation, after Pas- teur's hydrophobia method, of avian tubercle bacilli of graded and increasing virulence. These vaccinations were, however, frequently fatal to the animals, and the immunity obtained was but slight. Richet and Hericonrt have since claimed to produce complete immunity in dogs by intravenous inoculations of bird tubercle bacilli. These experimenters found that though harmless to the dog when first derived from the chicken, bird bacilli, by long cultivation in liquid media, become pathogenic for this animal, and by thus grading the virulence of the in- 282 IMMUNITY AND SERUM-THERAPY. jections complete immunity against any form of tubercu- lar infection Avas produced in tlie dog. As yet these strik- ing results have not been continued. The animals which I now present to you illustrate an attempt I have made along the same line to produce immunity in the rabbit. Cultures grown directly from the chicken's lesions in bouillon for, first, five weeks, then six months, were twice injected subcutaneously at intervals of twenty-one days in doses of 0.025 and 0.05, and a third injection of a still older culture was occasionally given. About one in four of the rabbits died within three months, profoundly ema- ciated, but without any visible tubercular lesions. The remaining animals recovered and were apparently in good health, when, together with an equal number of con- trols, they were inoculated in the anterior chamber. of the eye with cultures of Koch's bacillus derived from the tuberculous lesions of the rabbit, and cultivated about three months on glycerin agar. The results of these in- oculations ijresent many points of interest. In the con- trols, as is usually the case, if the operation has been done carefully and aseptically, and with a moderate amount of dilute virus, two days after the introduction of the virulent material in the eye little or no irritation is observed, and little is to be noticed for two weeks, when a steadil}^ increasing vascularity manifests itself, small tubercles appear on the iris, which gradually coalesce and become cheesy, intense iritis and general inflammation of the structures of the eye develop, the inoculation wound becomes cheesy, and in six to eight weeks the eye is more or less com^iletely destroyed and the inflammation begins to subside. The disease, however, remains generally localized in the eye for many months, and even perma- nently. In the vaccinated animals, on the contrary, the introduction of the virulent bacilli at once gives rise to a marked degree of irritation. On the second day the vessels of the conjunctiva are tortuous and enlarged, whitish specks of fibrinous-looking exudation appear in the iris and in the anterior chamber, and more or less in- TUBERCULOSIS. 283 tense iritis supervenes, but at the end of the second to the third week, when the eyes of the controls begin to sliow progressive and steadily increasing- evidence of iufiammatory reaction, the irritation in those of the vaccinated animals beg-ins slowly to subside and the eyes to mend. The vascularity^ is less, the whitish spots of fibrinous material appear smaller, the structures of the eye become clearer, the inoculation wound is but a bluish fibrous scar, until in from six to twelve weeks, in success- ful cases, all irritation has disappeared and the eyes present, as in the animals I now show you, but fibrous evidence of the traumatism and the inflammatory pro- cesses which have been set up by the inoculation. In all the controls, as you see, the inoculation wound is cheesy and the cornea and iris are more or less destroyed by tubercle and cheesy areas. " Some of the protected animals slowly relapse, and the one I now show you has small tubercles g-rowing on the iris ; but even in such eyes the entire absence of casea- tion is noticeable, and the disease progresses almost im- perceptibly. I have repeated this experiment on three sets of rabbits with about the same results each time. The vaccinations as practised are of themselves, in some instances, fatal, but the fact remains that where recovery takes place a marked degree of immunity has been ac- quired. I do not lay any claim, therefore, to have pro- duced a complete or permanent immunity by a safe method, but it seems to me that these eyes constitute a scientific demonstration of the fact that in rabbits pre- ventive inoculation of bird-tubercle bacilli can retard, and even abort, an otherwise progressive localized tuber- cular process so completely as to prevent destruction of the tissues threatened, and that the future study of anti- tubercular inoculation may not be as entirely hopeless as it has until recently appeared." We cannot attempt to review the extensive literature relatiuo- to the treatment of tuberculosis in man by 284 IM3IUNITY AND SERUM-THERAPY. means of Koch's tuberculin and the various preparations obtained from cultures of the tubercle bacillus. We may, however, briefly summarize the results reported by the statement that the clinical evidence shows that in early and properl3^ selected cases the treatment may exercise a favorable influence on the progress of pulmonary tuber- culosis ; but that it can by no means be accepted as a specific for this disease, and in advanced cases its inju- dicious use may do much harm. It is evident that in a disease in which recovery some- times occurs independently of treatment, and in clinical experiments upon patients who differ in their suscepti- bility to tubercular infection and in other important par- ticulars, and in the absence of " controls " such as we find it necessary to employ in experimenting- upon the lower animals, the reported results of any plan of treatment must be accepted with great caution. Having this in view we may refer to the very favorable report of Schiess-Bey and Kartulis (1893). Out of foi-tv- eight cases treated during a period of two years, sixteen are said to have been permanently cured. The authors referred to conclude from their experience that commenc- ing pulmonary tuberculosis may be surely cured by the use of tuberculin in the course of three to four months ; also that more advanced cases may be cured if the treat- ment is persisted in for six months to a year. Even cases having small cavities may, under exceptionally favorable conditions, be cured. But when large cavities exist, with hectic fever and night-sweats, the treatment is of no avail. The authors referred to consider the treatment as de- void of danger whf^n very small doses are employed at the outset, but these are not sufficient to effect a cure and must be gradually increased. Usually yV of a TUBERCULOSIS. 285 milligr. was given at lirst, but sometimes 1 milligi". was g-iven. These doses did not produce a decided reac- tion, and it was the aim of the authors to avoid such reac- tion by giving comparatively small doses at the outset and carefully increasing them. Finally, doses of 100 mil- ligr. were reached, after which the dose was again grad- ually diminished to 10 milligr., and subsequently in- creased again to the maximum. Of the 48 cases treated 33 were pulmonary and 13 " surgical " (tuberculosis of bones and skin). In the last-mentioned group of cases the results led the authors to formulate the following conclusions : " Certain forms of bone and joint tuberculo- sis, as well as gland tuberculosis, are cured more quickly by the use of tuberculin in combination with surgical measures than by surgical measures alone." Another conclusion which may indicate an important element in the success ascribed to the tuberculin treatment is that " the Egyptian climate is especially favorable for the tuberculin treatment." Escherich (1892) reports that in his experience pulmo- nary tuberculosis was rather unfavorably influenced by the tuberculin treatment than otherwise, and the results obtained in the treatment of tuberculous joints and glands could scarcely be considered favorable. But in the various forms of skin tuberculosis a curative effect was manifested. Klebs (1892) reports that out of 33 cases treated by himself with tuberculocidin (Klebs' s) 8 were cured and 21 improved. In 75 cases treated by his colleagues 6 were cured and 24 improved. He therefore concludes that this preparation is a very effective therapeutic agent for the treatment of tuberculosis. Kaatzer (1891) reports 44 cases of pulmonary tubercu- 286 IMMUNITY AND SERUM-THERAPY. losis treated. Of these 14 died, 16 were discliarg-ed cured (?), 9 improved under treatment, 4 did not improve, and 1 remained under treatment at the date of publica- tion. The commeneing- dose was from j\ to 1 milligr. ; the duration of treatment was from six to fifty - two weeks ; the average quantity of tuberculin administered to each patient 1,535 millig-r. In the last published report of the Adirondack Cottage Sanitarium (1894) Trudeau says : " The medical report continues to indicate that for a cer- tain class of cases the best and most permanent results are obtained by the addition of treatment by modified tubercu- lin to the usual climatic, hygienic, and open-air method." The use of tuberculin as a means of establishing the diagnosis of tuberculosis in cows, supposed to be infect- ed, has proved to be of considerable value, and it is now extensively employed for this purpose. Eber, in a sum- mary of the results reported by various authors up to the 15th of March, 1892, gives the following figures : In 446 tuberculous animals inoculated a positive reaction was obtained in 378 (84.75 per cent.). The dose given for a medium-sized animal was usually 0.4 to 0.5 c.c. of tuberculin diluted with nine to ten times its volume of carbolic acid water (0.5 per cent.). The injection was made in the side of the neck, preferably early in the morning or late in the evening. The characteristic reac- tion commenced in from six to eighteen hours after the inoculation, and lasted from three to twelve hours. The author named accepts an elevation from the normal tem- perature of 0.5° C, or more, lasting for several hours, as satisfactory evidence that the inoculated animal is in- fected with tuberculosis. TUBERCULOSIS. 287 Johne summarizes the reports of various veterinarians in Germany, made during- the year 1892. The total num- ber of animals was 287, the dose of tuberculin adminis- tered, from 0.1 to 0.5 gm. Of the inoculated animals 140 reacted and were slaughtered ; of these 121 were found at the autopsy to be tuberculous, and 19 were not ; 17 re- acted, but were not slaughtered ; 87 did not react, but were slaughtered, of these 82 proved not to be tubercu lous, and 5 (all reported by Schiitz, Rockl, and Lydtin) were tuberculous. Siedamgrotzky (1892) reports the results obtained in inoculations practised upon 259 cattle. Of these 209 re- acted with a temperature of 40° j3., or more, 17 with a temperature of 39.5° to 40° C, 37 with less than 39.5° C. In 197 the temperature reaction amounted to 1.5° C, or more, in 8 it was between 1 and 1.5° C, and in 54 less than 1° C. Those in the last g-roup were considered as free from tuberculosis, and the others as probably tuber- culous (197 as "very x^robably "). The results above referred to considered in connection with other published reports indicate that a reaction of V C, or more, is a pretty reliable indication that the in- oculated animal is tuberculous, althoug-h not an infallible test. Set'um-th€7'apy. Tizzoni and Cantanni (1892) have made experiments which lead them to the conclusion that the blood-serum of immunized guinea-pigs contains an antitoxin which may be successfully used in the treatment of infected animals of the same species, but their experiments can- not be accepted as conclusive and require confirmation, 288 IMMUNITY AND SERUM-THERAPY. especially in view of the fact that so many bacteriologists have reported their failure to establish immunity in these animals. Bernheim (1894), after immunizing animals by the injec- tion of sterilized cultures of the tubercle bacillus (treated for an hour and a half at 60° C. and then filtered), used blood-serum from these animals in the treatment of tuber- culosis in man. From 1 to 3 c.c. of this serum was in- jected every second day between the shoulders. The treatment in some cases was continued for five or six months. While the treatment did not jorove to be spe- cific, Bernheim considers the results obtained somewhat encouraging-. Quite recently (1895) Paquin has published a report of his method of obtaining an antitoxic serum and the re- sults of treatment in a number of cases of pulmonary tuberculosis. He obtains his serum from horses immu- nized by " laboratory processes " not described in detail. His conclusions are stated as follows : " 1. Sero-therapy in tuberculosis has proved, so far, efficacious. " 2. Blood - serum of horses seems naturally antago- nistic to the germs of tuberculosis, but cannot in its nat- ural state serve in treatment with much good, as it is too slow and it takes enormous quantities of it, too, to pro- duce useful results. " 3. Horse blood-serum may be rendered more strongly antagonistic to the tubercle germ by the treatment of the animals by a proper technique. " 4. A horse treated properly three months may yield serum with immunizing power that will jjrobably prove sufficient to arrest consumption in the first stages in three or four months, and sometimes less ; and in the second stage in four to six months or a year. TUBERCULOSIS. 289 " 5. Discrimination should be made in the selection of cases, and judgment exercised to exclude from the favorable class all those seriously complicated bacteri- olog'ically, certain cases complicated otherwise patho- logically, and other patients which practice alone will suggest. " 6. Inasmuch as hospitals for consumptives are needetl any way, the success has been sufficient already to war- rant the hope that in the near future such institutions shall be built on modern principles of hygiene, for the exclu- sive treatment, of tuberculosis. Sero-therapy, with the adjunct treatments, promises better success than ever ob- tained before. I do not designate this serum as a cure or a specific, but a valuable new remedy." BIBLIOGRAPHY. Bernheim, S. : Die Behaiidlung der Tuberkulose mit immunisiertem serum. Centralbl. f. Bakteriol., Bd. XV., p. 654, 1894. Braucaccio e Solaro : Osservazioni terapeutiche sul siero di sangue di caue nelle tuberculosi polmonare incurabili. Napoli, 1893, p. 377, 401. Bujwid, O. : La tuberculine, sa preparation, etc. Arch, des Sci. Biolog., St. Petersbourg, 1892, p. 243. Czaplewski, E., uud Roloff, F.: Beitrage zur Kenntniss der Tuberkii- linwirkung bei der Experimentellea Tuberkulose der Kaninchen und Meerschweincben. Berl. klin. Wchscbr., 1892, No. 29. Degive, A. : Le diagnose de la morve et de la tuberculose par les injec- tions hypodermiques de malleine et de tuberculine. Ann. de Med. veterin., xli., 247, 1892. De Scliweinitz : The Attenuated Bacillus Tuberculosis ; its Use in Pro- ducing Immunity to Tuberculosis in Guiuea-pigs. The Med. News, Philadelphia, 1894 (Dec. 8). Diem : Versuche mit Tuberculin bei Huhnertuberkulose. Monatssch. f. Prakt. Thierheilk., 111,481, 1892. Escherich, Th. : Die Resultate der Koch'schen Injectionen bei Scrophu- lose und Tuberkulose des Kinderalters. Jahrb. f. Kinderheilk., XXXIII., 369, 1892. Graraatschikoff, A. : Ueber die Wirkung des Koch'schen Mittels auf Tuberkulose Kaninchen. Arbeit a. d. Pathol. • Anatom. Inst, zu Tflbingen, 1892, Heft 3. 19 290 IMMUNITY AND SERU3I-TIIERAPY. Grancher et Martin : Etude sur hi vaccination tuherculeuse. Rev. de la Tuberculose, 1893, p. 289. Hammerschlag: Bakteriologisch-chepiische Uiitersuclmugen der Tuber- ivelbacillen. Sitzungsbericlit der K. Akad. der Wissensch. in Wien. , Dec. 13, 1888. Hericonrt, J., et Ricliet, Ch. : De la vaccination coutre la tuberculose liumaine par la tuberculose aviaire. Etudes exper. et Clin, sur la Tuberculose, iii., 365, 1892. Influence sur I'infection tuberculeuse de la transfusion du sang des chieus vaccines centre la tuberculose. Compte-rendu, Acad. des Sci., cxiv., 842, 1892. La Vaccination tuberculeuse chez le cbien. Le Bull. Med., 1892, pp. 741, 966. Helman, C. : Des proprietes de la tuberculiue provenant de bacilles tuberculeux cultives sur pommes de terres. Arcb. des Sci. Biolog. St. Petersbourg, t. 1, p. 140. Johne, A. : Tabellarische Zusamnienstellung der im J. 1892 mit Tu- berkujiu zu diaguostischeu Zwecken angestellten Impfversuche. Baumgarten's Jahresbericht, 1892, p. 676. Kaatzer, P. : Ueber das Koch'sclie Heilverfabren. Dtsclie. med. Wcbsckr.,1891. Kitasato, S. : Ueber die Tuberculin — Bebandlung tuberculoser Meer- schweinchen. Zeitschr. f. H3giene, XII., 321, 1892. Klebs, E. : Die Bebandlung der Tuberculose niit Tuberculocidin. Hamburg, 1892 (Voss). Klein, H. : Ursacben der Tuberculinwirkung. Bakteriologisch - ex- perimeutelle Untersuchungen, 8% 107 p., Wien u. Leipzig (Brau- maller), 1893. Koch : Weitere Mittheilung fiber das Tuberculin. Deutsche med. Woch., 1891, No. 43. Paquin, P. : Anti-tubercle Serum. Journ. Am. Med. Assn., vol. xxiv., 1895, p. 341. Pfuhl, E. : Beitrag zur Bebandlung tuberkuloser Meerscbweinchen mit Tuberculinum Kochii. Zeitschr. fur Hygiene, Bd. XL, 1892, p. 241. Sattler, H. : Ueber die Wirkung des Tuberculins auf die experimen- telle Tuberkulose beim Kaninclien. Dtsche. med. Wchschr., 1891, Nos. 1 u. 2. Schiess-Bey und Kartulis : Ueber die Resultate von 48 mit Tuberkulin behandelten Tuberkulosen. Zeitsch. fiir Hygiene, Bd. XV., p. 229. Siedamgrotzky, O. : Tuberkulinimpfungen grosserer Riuderbestande. Bericht. tlber d. Veterinarwesen i. Krg. Sachsen, 1891, p. 228. Tizzoni, G., und Cantanni, E. : Ueber das Vorhandensein eines gegen die Tuberculose Immunisirenden Princeps im Blnte von Tbieren welche nach der Metbode von Koch behandelt worden siud. Cen- tralbl. f. Bacteriol., XL, 82, 1893. TUBERCULOSIS. 291 Trudeau, E. L. : Ej^e Tuberculosis and Antitubercular Inoculation in the Rabbit. New York Medical Journal, 1893, vol. ii., p. 97. Yamagiva : Versucbi'esultate ilber die Wirkung des Tuberkulins auf die Impftuberkulose des Meerschweinchens und Kauinchens. Vir- chow's Archiv, CXXIX., Heft 2, 1892. XXI. TYPHOID FEVER. The bacillus discovered independently by Koch and by Eberth (1881) and carefully studied by Gaffky (1884) is now generally recognized as the specific cause of typhoid fever. It is constantly found in the form of small, scat- tered colonies, in the spleen, the liver, the glands of the mesentery, and the diseased intestinal glands in fatal cases of typhoid fever, and may be obtained, by puncture, from the spleen during life. The typhoid bacillus closely resembles the common bacillus found in the in- testine of healthy individuals which was first described by Escherich — Bacillus coli coTnmunis. But it can be differentiated from this bj^ certain biological tests and is generallj' recognized by bacteriologists as a distinct species. Both the typhoid bacillus and the " colon ba- cillus," however, vary considerably as a result of condi- tions relating to their environment, and it is difficult to determine whether certain bacilli of this group, some- times found in river-water, etc., are to be regarded as varieties of one or the other of the species mentioned or as distinct species. Brieger (1885) found in cultures of the typhoid bacillus small amounts of volatile fat acids, and when grape-sugar has been added to the culture medium lactic acid. He also obtained a highly alkaline basic substance pos- sessing toxic i^roperties which he named typhotoxin TYPHOID FEVER. 293 (C,H„NOJ. This he supposes to be the specific product to which the pathogenic action of the bacillus is due. It produces in mice and guinea-pigs salivation, paralysis, dilated pupils, diarrhoea, and death. More recent experiments by Pfeilfer (1894) lead him to conclude that the specific poison of the typhoid bacillus is not present in filtered cultures, but is closely associ- ated with the bacterial cells. According to Pfeifier the bacillus may be killed by a temperature of 54° C. without injury to this toxic substance. The fatal dose of the dead bacilli is from 3 to 4 milligr. per 100 gm. of body- weight, for guinea-pigs. Susceptible animals may be immunized by means of this toxic substance, and their blood is found to contain an antitoxin which has a spe- cific bactericidal action upon the typhoid bacillus. But, according to Pfeilfer, the blood-serum of animals immu- nized in this way does not differ from normal serum in its action on bacillus coli communis and other species of bacteria. These results are believed, by the author re- ferred to, to settle the question of the specific character of the typhoid bacillus, and to differentiate it from nearly allied species. The presence of a typhoid antitoxin in the blood-serum of individuals who have recently suffered an attack of typhoid fever has also been demonstrated by Pfeiffer. Chantemesse and Widal (1888) first showed by exj^eri- ment that susceptible animals could be made immune against the pathogenic action of this bacillus by the sub- cutaneous injection of sterilized cultures. Having found that four drops of a bouillon culture, three days old, in- jected into the j^eritoneal cavity of white mice caused the death of these animals within thirty-six hours, they pro- ceeded to inject small quantities (I c.c.) of a culture 294 IMMUNITY AND SERUM-THERAPY. which had. beeu sterilized by heat, and found that after several such protective inoculations the mice no longer succumbed to infection by an unsterilized culture. In experiments made upon rabbits, Bitter (1892) ar- rived at the conclusion that the immunity which he pro- duced in these animals by the intravenous injection of concentrated sterilized (by filtration) cultures was due to the presence of an antitoxin in the blood of the immune animals. Having- found that control animals were killed by intravenous injections of 1 c.c. of his concentrated so- lution of the products of the typhoid bacillus, he added to twice this amount of the toxic solution a certain quan- tity (?) of blood-serum from an immune rabbit, and in- jected the mixture into the circulation of rabbits with a negative result. Control experiments in which the toxic solution was mixed with blood-serum from non-immune animals showed that this had no antitoxic effect, and the animals died. Bruschettini obtained (1892) similar re- sults in his experiments upon rabbits with cultures ster- ilized by heat (60° C). He concludes from his experi- ments that the blood-serum of rabbits immunized in this way not only possesses antitoxic properties, but that it has g-reater g-ermicidal potency for the typhoid bacillus than the blood of normal rabbits. Stern (1892) has made experiments to determine whether the blood of recent convalescents from tj^phoid has greater germicidal power for the typhoid bacillus than that of other individuals. The result showed that the blood-serum from persons who had recently recov- ered from typhoid fever had no increased g"ermicidal power, but rather showed diminished potenc\' for the de- struction of typhoid bacilli. But blood from a man who had suffered an attack seventeen and a half years pre- TYPHOID FEVER. 295 vioiisly was found to liave unusual bactericidal power, altlioug-h it did not protect white mice from typhoid in- fection. On the other hand, blood from recent convales- cents served to immunize white mice, thus indicating the presence of an antitoxin. This is also shown by the ex- periments of Chantemesse and Widal (1892), who report their success in immunizing suscejitible animals by in- jecting the blood-serum of other animals previously made immune by repeated injections of sterilized (by heat) cultures. The authors last named have also tested the blood-serum of typhoid-fever patients, of recent con- valescents from the disease, and of persons who had suffered an attack some years before the experiment was made. The experiments were made upon guinea-pigs. Tlie authors conclude that " in general the guinea-pig is immunized against the action of virulent typhoid cult- ures by the subcutaneous injection of a small quantity of serum of persons who have suffered an attack of the dis- ease, no matter how remote." But this immunity was shown to be of short duration, and quite different from that induced by the injection of sterilized cultures, which does not immediately follow the introduction of the toxic substances, but requires a certain number of days for its development. The degree of immunity is said by the authors last named to depend to a considerable extent upon the dose given, and the animals treated in this way still resisted virvilent cultures at the end of two months. On the other hand, injections of blood-serum from im- mune individuals were effective in doses of a single c.c, within a few hours, and the immunity conferred had a comparatively brief duration. 296 IMMUNITY AND SEKUM-THERAPY. Seru7n-therapy. lu their experiments iu the treatment of infected rab- bits and guinea-pigs with serum from immune animals Chantemesse and Widal (1892) obtained favorable results when injections of 2 c.c. were made into the cavity of the abdomen within a few hours after infection. Favorable results were also obtained by injecting in the same way blood-serum from individuals who had suffered a recent or remote attack of the disease. Animals which recovered were immune against infection by the bacillus, but had no increased resistance against the toxic action of filtered cultures. In two cases in which typhoid patients in the eleventh and thirteenth day of the disease were treated with considerable doses of serum from immune animals, the progress of the disease was not arrested. Neisser (1893) by the injection of serum from a typhoid convalescent, fourteen days after the termination of the fever, was able to protect mice from three or four times the lethal dose of tyiihoid cultures. He injected 1 or 2 c.c. daily for four days. Stern (1894) has made similar and more numerous ex- periments upon mice and guinea-pigs, to test the anti- toxic power of the serum of individuals who had suffered an attack of typhoid fever. The inoculations were made in the peritoneal cavity in the proportion of from 1 to 1 to 10 to 1 of serum and culture. In guinea - pigs the serum was injected from sixteen to twenty -four hours before infection. Positive results were obtained with serum from 6 out of 8 recent convalescents, and with 3 out of 5 individuals who had suffered an attack from one to ten years previously ; and negative results with serum from two persons who had suffered 'attacks more than TYPHOID FEVER. 297 ten j'^ears before the experiment was made. In a series of experiments with blood-serum from fatal cases of typhoid this was found to be still more potent than the serum of recent convalescents. In doses of from 0.02 to 0.05 c.c. it protected guinea-pig's from lethal doses of a virulent typhoid culture injected after an interval of twenty hours. The blood was obtained from a vein an hour after the death of the patient. In a series of com- parative experiments with blood from individuals who had never had an attack of tyi3hoid a protective influence was shown to be exercised in some cases, but larger doses were required. Stern does not attempt to explain this fact, but suggests the possibility that persons whose blood-serum showed this antitoxic power may have at some time suffered a mild and unrecognized attack of typhoid fever. Cesaris-Demel and Orlandi (1894) have obtained results which confirm those above referred to as regards the pro- tective and therapeutic value of serum from animals im- munized against the typhoid bacillus ; and have obtained similar results in experiments with bacillus coli commu- nis. Moreover, they conclude, as a result of their exijeri- mental researches, that animals which have been im- munized for the " colon bacillus " furnish a serum which has protective and therapeutic value against infection by the tyiahoid bacillus, and the reverse. The serum from immunized animals has been used by the bacteriologists referred to in the treatment of typhoid fever in man, and the results are thought to be favorable, although the number of cases so treated is still too small to justify any definite conclusion. We may refer briefly, before concluding the present chapter, to recent experiments in the treatment of typhoid 298 IMMUNITY AND SKRUM-TIIERAPY. fever with sterilized cultures of the typhoid bacillus g-rowii in thymus bouillon (Friinkel) and of bacillus pyo- cyaneus (Rumpf). Frankel and Manchot (1893) treated fifty-seven cases of typhoid fever in the new general hos- pital at Hamburg-Eppendorf with cultures of the typhoid bacillus in thymus bouillon, Avhicli had grown for three days in an incubating oven at 37" C, and were then sterilized by exposure to a temperature of 60° C. The treatment was commenced by the subcutaneous injection of 0.5 c.c. of the sterilized culture ; the following day a dose of 1 c.c. was given ; after this the injection was re- peated every second day, and the dose was increased each time by 1 c.c. Usually there was a noticeable rise of temperature as a result of the injection, and in some cases a more or less jaronounced chill. On the third day of treatment there was usually a fall in the temperature, followed by a still greater reduction on the following- day. The conclusion is reached that the course of the fever is materially influenced by the treatment, that it is changed from a continued to a more or less remittent character, and that complete apyrexia occurs in a shorter time. Rumpf (1893) was induced to test the value of steril- ized cultures of Bacillus jDyocyaneus, because it had been shown by experiments upon animals that such sterilized cultures exercised an immunizing influence against ty- phoid infection. He followed Friinkel's method, giving on the first day 0.5 c.c. of the sterilized culture, on the second day 1 c.c, on the fourth day 2 c.c, the sixth day 4 c.c, the eighth day 6 c.c. The injections were made in the gluteal region. They were commonly followed by a rise in the temperature and a subsequent fall, with dimin- ished pulse-rate and profuse perspiration. Sometimes a TYPHOID FEVER. 299 chill and a rise of temperature followed the injection. No effect was observed upon the secretion of urine or the respiration, but, as a rule, a general improvement in the feeling of the iDatient and a prompt disappearance of de- lirium, when it existed, was noted. Thirty cases were treated, with two deaths, one from pneumonia and one from intestinal hemorrhage. In a recent communication (1894) Kraus and Buswell have reported the results of additional experiments. They treated in all twelve cases with sterilized pyocya- neus cultures ; ten recovered, and two died. As a rule, the immediate result of an injection was a reduction of temperature lasting for several hoiirs. It was found also that an increase in the number of leucocytes in the blood followed the injections, and the same result occurred when the injections of sterilized pyocyaneus cultures were made in rabbits. The conclusion is reached, how- ever, that this method of treatment has no specific cura- tive value. BIBLIOGRAPHY. Bitter, H. : Ueber Festigung von Versuchsthieren gegen die Toxine der Typhusbacillen. Zeitschr. f. Hygiene, XII., 298, 1892. Cesaris-Demel »nd Oiiandi : Die Serunitherapie und das Bacterium Coli. Abst. in Centralbl. fur Bakteriol., XVI., 246, 1894. Chantemesse et Widal : De Timmunite contre le virus de la fievre ty- phoide conferee par les substances solubles. Ann. de I'lnst. Pas- teur, t. ii., 1888, p. 54. Etude experinientale sur I'exaltation, Timmunisation. et la thera- peutique de I'infection typhique. Annal. de I'lnst. Pasteur, 1892, p. 755. Frankel, E. : Ueber spezitisclie Bebandlung des Abdominallyphus. Dtscli. nied. Wchschr., 1893, No. 41. Kraus, Fr. , u. Buswell, H. C. : TJeber die Bebandlung des Typbus ab- doniinalis mit abgetoteten Pyocyauus - Kulturen. Wien. Win. Wcbscbr., 1894, p. 511, 595. 300 IMMUNITY AND SEIIUM-THERAPY. Montcfusco, H. : Contributo alia l)iologia del bacillo del tifo. La Rif. Med., 1893, p. 155. Neisser : Zeitscbr. fUr klin. Med., 1893, Bd. XXII. Petruschky, J. : Ueber die Ait der Patbogenen Wirkung des Typbiis- bacillus a\rf Tbiere und Uber die Verleibung des Impfscbutzes gegen dieselbe. Zeitscbr. f. Hygiene, XII., 261, 1892. Pfeiffer, R. : Ueber die specifiscbe Immunitatsreaction der Typbu.sba- cillen. Vorlaufige Mittbeilung. Deutscbe raed. Wcbscbr., 1894, No. 48, p. 898. Riimpf, Tb. : Die Bebandlung des Typbus xVbdominalis mit Abgetote- ten Kulturen des Bac. Pyocyaneus. Dtscbe. med. Wcbscbr., 1893, p. 987. Stern, R. : Ueber Immunitat gegen Abdominaltyplms. Dtscbe. nied. Wcbscbr., 1892, No. 37. Ueber die Wirkung des Menscblicben Blutserums auf die Expe- rimeutelle Typbusiat'ectiou. Zeitscbr. fUr Hyg., XVI., 458, 1894. xxn. YELLOAV FEVER. In the writer's report (1890) upon the results of his in- vestigations relating- to the etiology of yellow fever, his conclusions are stated as follows : " The experimental data recorded in this report show that — " The specific infectious agent m yellow fever has not been demonstrated. The most approved bacteriological methods fail to demonstrate the constant presence of any particular microorganism in the blood and tissues of yellow-fever cadavers. " The microorganisms which are sometimes obtained in cultures from the blood and tissues are present in com- paratively small numbers, and the one most frequently found {Bacterimn coli communis) is present in the intes- tine of healthy individuals, and consequently its occa- sional presence cannot have any etiological import. " A few scattered bacilli are present in the liver, and probably in other organs, at the moment of death. This is shown by preserving portions of liver, obtained at a recent autopsy, in an antiseptic wrapping. " At the end of twenty -four to forty-eight hours the in- terior of a piece of liver so preserved contains a large number of bacilli of various species, the most abundant being those heretofore mentioned as occasionally found in the fresh liver-tissue, viz., BaderiuTn coli comirmnis and Bacxllxis cadaveris. " Blood, urine, and crushed liver-tissue obtained from a 302 IMMUNITY AND SERUM-THERAPY. recent autopsj' are not patliog-enic, in moderate amounts, for rabbits or guinea-pigs. " Having- failed to demonstrate the presence of a specific ' germ ' in the blood and tissues, it seems probable that it is to be found in the alimentary canal, as is the case in cholera. But the extended rtesearches made and recorded in the present report show that the contents of the intes- tines of yellow-fever cases contain a great variety of ba- cilli and not a nearly pure culture of a single species, as is the case in recent and tyj)ical cases of cholera." At the time of my visit to Brazil (1887) Dr. Domingos Freire iDresented me with a culture of a micrococcus which he, at that time, considered the specific yellow- fever germ. This micrococcus did not at all conform with the descriptions which he had repeatedly published of his " cryptococcus xanthogenicus." It did not pro- duce pigment, either black or yellow, was not pathogenic for small animals, except when injected in very large doses, and gave rise to no symptoms in inoculated ani- mals which could be identified with those of yellow fever. On the contrary, it was an ordinary stapholycoc- cus, which corresponded in its morphology and growth in culture media with the well-known staphyloccus pyo- genes albus. With reference to this micrococcus I quote from my report above referred to as follows : " That this micrococcus bears no relation to the etiol- ogy of yellow fever is fully proved by my extended cult- ure experiments in Havana during the summers of 1888 and 1889. In the entire series of autopsies I have made cultures from the liver, and in a considerable number from blood obtained directly from the heart, and I have not obtained this micrococcus of Freire in a single in- stance, although the culture medium commonly employed — flesh peptone-gelatine — is a very favorable one for the YELLOW FEVER. 303 growth of this coccus. Nor has it been found iu the ex- tended series of sections which I have made from the liver and kidney preserved in alcohol from my Havana autopsies. In one case, only (Case X., 1888) I have found micrococci in sections from the kidney, but as the micro- coccus of Friere, in its form and dimensions, resembles many others, it is impossible to say that this is or not the Freire coccus. The finding- of micrococci in this case, however, does not invalidate the general result, which is that micrococci are not found hi tJie Mood and tissues of yel- low fever cadavers.'''' In a paper read at the Quarantine Conference, held in Montg-omery, Ala., in March, 1889, the present writer says : " I may say before g-oing- any further that my faith in a living- infectious agent as the specific cause of this dis- ease is by no means diminished by my failure thus far to demonstrate the exact form and nature of this lij potheti- cal ' g-erm.' The present state of knowledge with refer- ence to the etiolog-y of infectious diseases in g'eneral, and well kno^vn facts relating to the origin and spread of yel- low-fever epidemics fully justifies such a belief. The a priori grounds for such faith I stated as long ago as 1873, in a paper published in the American Journal of the 3£edi- ccd Sciences (July, 1873) ; and the progress of knowledge since that date has all been in the direction of support- ing this a 2^riori reasoning. But yelloAv fever is by no ' means the only infectious disease in which satisfactory evidence of the existence of a living infectious agent is still wanting. In the eruptive fevers generally no dem- onstration has been made of the specific etiological agent — at least none which has been accepted by compe- tent pathologists and bacteriologists. Again, in the in- fectious disease of cattle known as pleuro-pneumonia, notwithstanding very extended researches by competent investigators in various parts of the world, no satisfac- 304 IMMUNITY AND SERUM-THEEAPY. tory demonstration of the germ lias been made. Tlie same is true of hydrophobia, in which disease we are able to say with confidence the infectious agent is pres- ent in the brain and spinal cord of animals which suc- cumb to rabies ; this infectious agent is destroyed by a temperature which is fatal to known pathogenic microorganisms (65° C), and by various germicidal agents, yet all efforts to cultivate it or to demonstrate its presence in the infectious material by staining processes and microscopical examination have thus far been un- successful." In a paper read before the College of Physicians in Philadelphia, in 1888, I give the following account of my investigations with reference to the inoculations practised by Dr. Domingos Freire in Brazil : " Facts relating to the endemic and epidemic preva- lence of yellow fever, considered in connection with the present state of knowledge concerning the etiology of other infectious diseases, justify the belief that yellow fever is due to a living microorganism, capable of devel- opment, under favorable local and meteorological con- ditions, external to the human body, and of establishing new centres of infection when transported to distant local- ities. " Inasmuch as a single attack of yellow fever, however mild, protects, as a rule, from future attacks, there is reason to hope that similar protection would result if a method could be discovered of inducing a mild attack of the disease by inoculation or otherwise. . . . " My own researches, recorded in the foregoing report, show that no such microorganism as Dr. Domingos Freire, of Brazil, has described in his published works, or as he presented to me as his yellow-fever germ at the time of my visit to Brazil, is found, as he asserts, in the blood and tissues of typical cases of yellow fever. " There is no satisfactory evidence that the method of YELLOW FEVER. 305 inoculation practised by Dr. Domingos Freire has any prophylactic value. "Dr. Freire' s Protective Inoculations. — Having demon- strated that Dr. Freire's claim to have discovered the specific cause of yellow fever is without scientific founda- tion, it may be thought that no further demonstration is required in order to show that his x^reventive inoculations are without value ; for these inoculations are said to have been made with cultures containing the attenuated mi- crobe of yellow fever. These inoculations have, however, been made upon so large a scale, and the statistical re- sults, as presented by Dr. Freire, appear so favorable to his method that it becomes necessary to analyze these statistics : and if, as he claims, they establish the fact that the mortality from yellow fever is very much less among those who have been inoculated by him than among non-inoculated persons exposed in the same way, we shall be obliged to concede the value of his method, although the rationale of the protective influence may not be apparent. In my detailed report I have reviewed at length Dr. Freire's statistics in the light of the facts developed by my personal researches in the city of Rio de Janeiro, where the inoculations were made. I cannot attempt to bring the evidence before j^ou at the present time, and I have already stated to you my conclusion with reference to the matter. In support of this conclu- sion I shall, however, quote a few extracts from my report. "In 1884 Dr. Freire inoculated 418 persons, Avhose names, ages, place of residence, and length of residence in Brazil are given in an appendix to his principal work, published in 1885. In regard to the evidence afforded by these inoculations, I have written as follows : " Dr. Freire admits that ' during the epidemic season a great number of the vaccinated were attacked by the mal- ady,' but claims that these attacks were of a mild char- acter, yet he gives us the names of seven vaccinated per- sons who died from the disease. This list has been added to by some of Dr. Freire's confreres, as will be seen 20 306 IMMUNITY AND SERUM-THERAPY. by the following- translation of a letter published in one of the newspapers of liio, and bearing- date, May 5, 1887. This letter is sig-ned by Dr. Aran jo Goes, at present a member of the Central Board of Health, and a g-entleman whose statements are worthy of the fullest confidence : " ' My letter to the Imperial Academy of Medicine hav- ing been published, it now behooves me to publish the statistics relating to the vaccinations on Morro de Yiuva. " ' One fact seems to me to be definitely demonstrated, that is the icorthlessness of Dr. Freire's vaccinaiion, as is well known to the medical profession of this city. " ' A year ago I wrote the following : " ' The want of skill which he displayed in his first ex- periments, the false conclusions which he has drawn therefrom, and the tliougiitless precipitation with which he has hastened to make known incomplete results with- out accompanjdng them with a single qualifying: remark vitiate all the methods to wdiicli he may hereafter resoi-t to corroborate his statements {Journal do Coinmerc'io, April 20, 1883). " ' The mortality among the persons vaccinated on Morro da Viuva furnishes one more proof that I was rig-ht in saying- this, as I now proceed to demonstrate, " ' There were vaccinated in this district 60 persons. " ' Sixteen removed shortly after the commencement of the epidemic, and 44 remained exposed to its influence. Of these 22 had yellow fever, 9 of whom died. . . .' " In 1885 Dr. Freire resumed his inoculations on a larger scale-, but instead of selecting unacclimated strangers, those inoculated w^ere for the most part natives of Brazil, or Portuguese who had lived for a number of years in Rio and who had passed through one or more epidemics. A considerable number of negroes were also inoculated and included in the statistical tables. With reference to Dr. Freire's statistics for the year 1885 I quote from my report as follows : " ' Dr. Freire has omitted to state one very important fact with reference to the vaccinations practised during YELLOAY FEVER. 307 the period included in this tabular statement. The date of the vaccinations is not given. Fortunately I am able to supply this omission from his journal containing- the names of the vaccinated, which he kindly placed in my hands during my stay in Rio. I find from this record that the inoculations were practised as follows : January 392 February 342 March 611 April 189 May 273 June 813 July 481 " ' Now it is well known that June and July are months during which 3^ellow fever does not jarevail in Eio, and that, in fact, the month of May furnishes, as a rule, but few cases. " ' The exposure even in an epidemic year amounts to very little during the months of May, June, and July, and may be considered practically nil in a year like 1885, when the whole mortality was only 278 in a city of 400,- 000 inhabitants. But Dr. Freire has included in his list 1,294 persons who were vaccinated during- the healthy winter months of June and July, and who presumably had been exposed during- the preceding comparatively unhealthy months of January, February, March, and April. If these 1,291 individuals were protected from an attack of yellow fever by the inoculation practised in June or July, what protected them from being- attacked during- the preceding months when yellow fever was pre- vailing to some extent ? . . .' " I pass now to the year 1886, during- which Dr. Freire inoculated 2,763 Brazilians and 710 foreig-ners, again in- cluding in his statistical tables those vaccinated after the epidemic season had passed. In reviewing- these statis- tics I remark as follows : " ' We have quoted this last report of Dr. Freire in ex- tenso in order to do him full justice by allowing- him to 308 IMMUNITY AND SERUM-THERAPY. state his own case. We shall now proceed to show that his statistics are fallacious, and that the percentage of mortality among the vaccinated, which he finds to be ten times less than among the non-vaccinated, results from a misuse of the statistical method and from a number of factors which are favorable to Dr. Freire's statistics as he has stated them, but not to a fair test of his method of prophylaxis. " ' In the first place, we would call attent/on to the fact that while during- the comparatively healthy year, 1885, the immunity among the vaccinated of that year is said to be complete (see report of 1885), the number of deaths during the epidemic year which followed is stated by Dr. Freire himself to have been eight. Taking all of the vaccinated of the two years, and without making any allowance for the considerable number of persons vacci- nated who had, no doubt, left the city before the epidemic of 1886 occurred, Dr. Freire, with a total of 6,524 vacci- nated, and a total of 8 deaths, makes the proportion one per thousand. This is equivalent at the outset to an ad- dition of 1,476 persons to the number vaccinated, who being imaginary persons and not having been exposed to the epidemic influence simply aid in rounding up the general percentage of mortality in Dr. Freire's favor to the even figure of one per thousand. This is but one of many factors which go to make this favorable showing. Reference to Dr. Freire's MS. journals, which he kindly placed in my hands, shows that of the total number vac- cinated during the two years, 4,465 were vaccinated prior to the epidemic of 1886 ; that is to say, before the 1st of January, 1886. How many of these left the city before the outbreak of the epidemic, how manj'^ were only temporarily in the city when vaccinated, how many died from other diseases I cannot say ; but it is a signifi- cant fact that of the 3,051 vaccinated prior to August, 1885, Dr. Freire has only one fatal case to report, while out of 460 persons vaccinated in January and February, 1886, he reports 5 deaths, a mortality of more than one YELLOAY FEVER. 309 per cent., wliicli he gives as tlie general mortality among the non-vacciuated. This is not apparent from his own statement of the case, but is nevertheless true, as I shall proceed to show. In his report, which we have just given in full, he does not give the date of the vaccination of these individuals, but upon referring to his MS. jour- nals for 1886 I find that No. 3 of his list, Jose, son of Jose da Costa Vieira, was vaccinated February 12, 1886 ; No. 4, Paschoal Ruffino, on the 6th of February, 1886 ; No. 5, Henri Constance, on the 1st of January, 1886 ; No, 6, Fernando Argenteiro, on the 20tli of February, 1886 ; and No. 7, Antonio Saraiva, on the 12th of February, 1886. The same MS. record for 1886 shows that during these two months — January and February, 1886 — the total num- ber vaccinated by Dr. Freire was 460. That is to saj;-, the mortality among those vaccinated during these two months was more than one per cent. On referring to the mortality list of the city for the same two mouths I find the total number of deaths to have been 369, which in a total susceptible population of 160,000 (Dr. Freire's esti- mate) would give a mortality of 1 in 436.' " Dr. Carl Seidl, of Rio de Janeiro, wlio is Director of the San Sebastian Yellow Fever Hospital in that city, in a recently (1894) published communication calls attention to the fact that Dr. Freire's yellow-fever vaccinations " have been in progress since 1883 and yelloAv fever con- tinues, becoming year by year more formidable and de- structive, instead of being graduallj'^ stamped out as was prophesied by Freire. Statistics will never convince the bacteriologists of the world, the medical fraternity of Brazil, nor the public, of the value of these vaccinations. For where is the physician, where is the intelligent man in this town, who does not know cases and cases in which said vaccination was not only useless but perhaps harm- ful because it caused a false confidence ? In the labora- 810 IMMUNITY AND SEKUM-TIIERAPY. fcories of Paris, Vienna, and Berlin are long- series of pathogenic germs, some only recently announced, yet there is no culture of the xanthococcus which has been discovered for so many years and i^ut to practical account in Kio." * The evidence in favor of the value of the protective in- oculations practised by the method proposed by Profes- sor Carmona (1881), of the City of Mexico, is not satisfac- tory, and this method was soon abandoned. It consisted in the subcutaneous injection of material obtained by desiccating the urine of yellow-fever patients freely ex- posed to the air in shallow vessels. Carmona says, with reference to his inoculations : " I usually place one or two centigrammes of the drj' residue in a gramme of distilled water, I triturate it in such a manner that the mixture is as perfect as possible, and charging a Pravaz syringe, I make a subcutaneous injection in the right arm. The results are various, but no serious accident has ever occurred. " I count, to-day, nearly two hundred persons inocu- lated, and among them several experienced, some hours after the inoculation, a febrile movement, which some- times caused the thermometer to mount to 38.5° C. The duration of this febrile movement did not exceed twenty- four hours. The local accidents have been most varied. There was almost always tumefaction at the point of in- oculation, but the extent and size of this tumefaction varied greatly. In many cases there was redness of the skin. These local phenomena lasted four or five days, but, in general, those inoculated continued about their ordina- ry affairs. Once only I have seen developed a phlegmon, which terminated by sui3puration." * Quoted from an editorial article in the Journal of the American Medical As- sociation of January 5, 1895. YELLOW FEVEK. 311 The " mosquito inoculations " of Dr. Carlos Finlay, of Havana, have not g-iven results which have induced any of his medical confreres to adopt his method ; and the facts, as stated by himself, do not appear to give any support to the supposition that these inoculations have a protective value. The method consists in allowing- a mos- quito to fill itself with blood from a yellow-fever patient, and, after two or three days, when it has digested this meal, applying it to the susceptible individual, who is supposed to be inoculated when the insect introduces its sting for the purpose of filling- itself with blood. But there seems slig-ht chance that such inoculation would occur even if the infectious element had been proved to be present in the blood drawn from the yellow-fever pa- tient. For this blood is dig-ested and passes through the alimentary canal, and the lancet and sucking" apparatus of the insect would probably be pretty well cleaned dur- ing- the interval between its application to the yellow- fever case and the person to be protected. Dr. Finlay says in his last published paper (1894) : " In my inoculation experiments the interval between the application of the contaminated mosquito to a suscep- tible person and the appearance of the first symptoms of a mild attack of the disease (when such a one did occur) has varied between five and twenty-five days, the latter term being the one that I have fixed upon, beyond which any morbid symptoms would be considered as indepen- dent of the inoculation. In the majority of cases no pathogenic effects were produced." It is well established that the period of incubation in yellow fever is short, usually, if not always, less than five days ; and there is not the slightest reason for the assump- tion that the so-called mosquito inoculation had anything 812 IMMUNITY AND SERUM-THERAPY. to do with the " acclimation fever " which a small propor- tion of those inoculated sufltered in from five to twenty - five daj'^s after this operation. All of the inoculated remained in Havana and were exposed to the same in- fiuences which produce the so-called " acclimation fever" in other individuals not inoculated. Dr. Finlay states his results as follow's : " Among" the eighty-seven who have been under obser- vation the following- results have been recorded : " 1. Within a term of days, varying" between five and twenty-five after the inoculation, one presented a mild al- buminuric attack, and tldrteen only 'acclimation fevers." " 2. Among" the seventy-three who did not present an 3" distinct attack within the first twenty-five days — 40 were subsequently attacked with simple ' acclimation fever.' 5 suffered an attack of regular albuminuric yellow fever. 4 had melano-albuminuric yellow fever. 24 have never had any fever of the yellow-fever type. 73 " Tioo of the melano-albuminuric, and one of the albu- minuric, were fatal, giving" a mortality of 3.87, equivalent to 3| per cent. One occurred in 1884, and the two others in 1893." The most significant fact in this statement is that nine of those inoculated suffered attacks of yellow fever- three of which were fatal. The fact that twenty-four out of the eighty-seven inoculated have remained in Havana without suffering an attack of yellow fever cannot be ac- cepted as evidence that they are protected by the mos quito inoculation practised by Dr. Finlay, for it is well YELLOW FEVER. 313 known in HaNtuia that strangers wlio have not been inoc- ulated often remain in that city for many years without having- yellow fever. Moreover, the forty cases of " ac- climation fever," which, according to Dr. Finlay, occurred among the inoculated, belong in the same category as the thirteen cases which occurred in from five to twenty- live days after the inoculation was practised. This twenty-five-day limit is entirelj^ arbitrary, and it is diffi- cult to see how Dr. Finlay can attribute the so-called " acclimation fever," which occurs in less than twenty- five days, to the direct effects of his mosquito inocula- tions, and cases which occiir after twenty-five days to a protective influence exercised by these inoculations. As a matter of historical interest in connection with the subject of protective inoculations against yellow fever, I introduce here an account of the inoculations made in 1854 and 1855 by Dr. William Lambert de Hum- boldt, and those made in 1864 by Drs. Lebredo and Cis- neros, of Havana. Dr. de Humboldt claimed to be a nephew of the cele- brated Alexander v. Humboldt. He asserted that he had discovered a sure means of protecting from yellow f-ever by inoculations with the venom of a poisonous snake, found in Mexico, but kept as a secret the precise species of snake from which his material for inoculations was ob- tained. His theory was based upon the fact that symp- toms somewhat resembling those of yellow fever are pro- duced by the venom of certain poisonous snakes. These sj^mptoms are hemorrhage from the gums, slow pulse, fever, etc. The first experiments of Humboldt were made in Vera Cruz, in 1847, by authority of the govern- ment, upon condemned prisoners. According to Boudin the matter inoculated was an ounce of sheep's liver 314 IMMUNITY "and SEllUM-TIIERAPY. wliicli had been bitten l>y six of these ijoisonous ser- pents. This was left to undergo putrefaction before it was used for the inoculations. A history of Humboklt's inoculations has been written by Dr. Nicolas B. L. Manzini. This is a volume of 240 pages, which was published in Paris in 1858. I shall quote from this work, which is entitled " Histoire de f inoculation 2^^'eservatiue de la Jievre jaune, prat'tqnee pur ordre du gouvemement espagnol, a Vhopital Jiul'datre de la Havane. Hedlgce par Nicolas B. L. Manzini, docteur en medecine de la, Facidte de Paris, mernhre titulaire de la Societe mediccde d' emulation de Paris, medecin de V Associ- ation de hienfaisarvce frangaise de la Havane.'" " I. In the month of October, 1854, Dr. William Lambert de Humboldt, residing then in New Orleans, wrote to General Don Jose de la Concha, Governor of the island of Cuba, announcing to him that he had discovered a sub- stance, the active principle of which was the venom of an ophidian, which substance, inoculated by vaccination in persons who were strangers in the localities where j^ellow fever reigns as an epidemic, protected them from this terrible malady. M. de Humboldt said that during a period of nine years he had inoculated fourteen hundred and hftj^-eight individuals. Of those whose history he had been able to follow he had only seen seven attacked with yellow fever, and of these only two had died. Out of three hundred and eighty-six inoculated in New Or- leans he had not in any case seen yellow fever character- ized by the pathognomonic symptoms — black vomit, etc. Finally, M. de Humboldt offered to apply his preserva- tive to the Spanish troops of the island of Cuba, in a most disinterested manner. "II. General Concha first consulted Dr. Basterreche, chief of the corps of military sanitation of the island of Cuba, with reference to this important affair, who consid- YELLOW FEVER. 315 ered it prudent to consult with some of his medical friends, who all g-ave a favorable opinion. Then General Ooncha, who at the outset took a lively interest in the question, consulted, officially, the University, submitting- at the same time a memoir by M. de Humboldt relative to it. This corporation judged that the experiment Avas admissible and that the facts would decide the question. In consequence of this decision M. de Humboldt was invited to come to Havana. A ward in the military hos- pital w^as placed under his absolute direction. It was likewise agreed that all of the inoculated who should sub- sequently fall sick should be placed in his care, and that he could be assisted or reiDlaced by persons of his selec- tion. A commission of tire University was named to fol- low the march of the operations and to make exact obser- vations. It Avas composed of Drs. Cowley, Castroverde, and Benjnmeda. " III. M. de Humboldt had scarcely arrived in Havana Avhen I put myself in relations with him. He appears to lie thirty-five to thirty-six years old. He is blond, high, and slender. His chest, flattened in front, offers the con- formation peculiar to the tuberculous. . . . Although his health is precarious he is endowed with a feverish activity and with a resistance to work which is surpris- ing. M. de Humboldt speaks four languag-es, among them the Spanish and French, as perfectly as is possible for a foreigner. German is his native tong-ue, and he speaks, besides, English. . . . " The special history of the phenomena of the inocula- tion which we are about to write is derived from seventy- nine observations collected and written out by M. de Humboldt and myself. Besides, we have drawn some in- formation relative to the circulation and headache from one hundred and thirteen other cases, collected in the military hospital under the direction of M. de Humboldt. " We have little to say of the character of the substance inoculated, which has precisely the appearance and the odor of the liquid residue of animal putrefaction. 316 IMMUNITY AND SERUM-THERAPY. 1. LOCAL PHENOMENA OF THE INOCITLATION. " As soon as the iiiocnlatioii is made, a crossed bandage is applied to the puncture and no further attention is paid to it. If it is examined at the end of a few moments it will be found to be surrounded with an elevation in the form of a white papule, diaphanous, and quite analogous to that produced by the puncture of a bed-bug. This phenomenon was visible at the end of five minutes or even of three. It was no longer seen at the end of twenty- four hours, of twelve, or even of ten. A sensation of tin- gling and numbness, a veritable phenomenon of slight an- esthesia, soon manifested itself in the forearm and lasted for a variable time ; we have seen it joersist until the fourth day in the case of Madame Mercedes Parodi. We have never seen any swelling of the axillary glands. 2. OUTLINE OF THE SICKNESS INDUCED. " Syncope may occur at the moment of inoculation — this soon i:)asses — or a nervous trembling, which is more rare but which lasts longer. The pulse is accelerated under the influence of the emotion of the moment. At the end of seven hours (all of the times which follow are the mean time deduced from the extremes) the pulse is modified in a permanent manner ; it is more frequent or slower, stronger or more feeble. At the end of eleven hours there is febrile heat ; at the end of fourteen, head- ache, thirst, loss of appetite ; at the end of sixteen the face is red, the conjunctivae injected, laclirymation. A commencement of swelling of the gums is observed and slight colicky pains are experienced, produced by the medicine which the patient commenced to take immedi- ately after the inoculation. " First. At the end of eighteen hours pain in the gums, which commence to be colored around the borders of the teeth ; pain in the salivary glands and in the direction of the different nervous branches of the face and cranium. YELLOW FEVER. 317 Second : of nineteen hours, pains in the lower jaw in the direction of the inferior maxillary nerve, lassitude ; of twenty hours, bitter taste, somnolence, oedema of the face ; of twenty-two hours, feeling- of constriction of the throat, without visible modification of the mucous membrane ; of twenty-three hours, yellowness of the skin ; of twenty- four hours, hemorrhag"e from the gums ; of twenty-eig-ht hours, eyes yellow, chills ; of twenty-nine hours, inflam- mation of tonsils ; of thirty hours, pain in reg-ion of kidneys ; of thirty-six hours, oedema of eyelids ; of thirty- eight hours, muscular and articular pains ; of forty hours, toothache ; of seventy-two hours, oedema of the lower lip ; at different times, erotic phenomena. During conva- lescence, cutaneous itching ; cutaneous eruptions of dif- ferent kinds. 3. IMMEDIATE TREATMENT. " As soon as the inoculation was made we administered a sirup composed as follows : ^ . Sirop. de mikania giiaco 187 gm. Sirop. de rhubarbe 125 gm. lodure de potassium 4 gm. Gomme gutte 12 gm. D. et M. " This sirup was administered in the following- manner : First day, a tablespoonful every two hours ; second day, one every four hours ; third day, one morning- and even- ing. If the symptoms are more violent, the interval is shortened ; and if that is not sufficient, it is necessary to add to the sirup an infusion of mikania, a teacupful every two hours." We shall not attempt to follow our author in his ac- count of the symptoms produced by the poisons of the various venomous snakes and in his attempt to show a resemblance between these symptoms and those of yellow fever. Nor shall we quote his account of the results of 318 IMMUNITY AND SERT M-TIIERAPY. the inoculations made, inasmuch as we have these from an official source in the report of Dr. Bastan-eche, which, with great fairness, Dr. Manzini has included in his vol- ume, although he insists, contrary to the opinion and fig- ures of Dr. Bastarreche, that the inoculations were at- tended with a comparatively favorable result. It will be sufficient to introduce here a sing-le one of the tables appended to the report of Dr. Bastarreche. TABLE NO. V. — COMPARATrVE SUMMARY. Not IxoctXATED. ISOCCI-ATED. Ratio of Attacked. Died. Ratio. Number. Attacked. Died. Murtality. Army Navy 1,045 264 254 24.31 47 i 17.80 1 1,214 1,263 84 144 21 46 25.00 31.94 Total.... 1,309 ! 301 22.99 2,477 228 67 29.39 Nothing further has been heard of Dr. Humboldt's method of inoculation in Havana, and the inference is that by common consent the experiment made on so large a scale, and under such favorable conditions, is regarded as having demonstrated its inutility. Another attempt to protect from yellow fever by inocu- lations is that made in 1864 by two physicians of Havana, members of the Academy of Sciences, and recorded in the " Anales " of the Academy. "W'e quote the translation which Dr. Stanford E. Chaille, President of the Havana Yellow Fever Commission of the National Board of Health, has introduced into his elaborate and valuable " Keport : " * * Annual Report of the Natioual Board of Health for 1880, p. 160. YELLOW FEVER. 319 " In June, 1864, Drs. Lebredo and Cisneros, members of the Academy, and distinguished physicians, tested the prophylactic value of inoculated dew, by request of Drs. Masnata and Fraschieri, who had claimed for it protec- tive power. " The substance used was not, as had been generally supposed, natural dew, but an artificial dew obtained by the condensation of vapor of the atmosphere of the closed room of a yellow-fever patient, and collected on the sur- face of bottles containing water of a lower temperature than that of the surrounding- air. After jirolonged ex- amination the following were our conclusions : " Yellow fever is not a contagious nor an inoculable disease, hence the inoculation of dew cannot be effec- tive. There is no such entity as the so-called ' fever of acclimation,' audit has not been proved that the ailments thiis designated protect from yellow fever. The symp- toms following the inoculation of ' rocio ' lack the uni- formity necessary to constitute a classifiable pathologi- cal condition as dependent solely on the inoculation ; the very slight intensity of the phenomena discredit their identity with those of the so-called ' fever of acclima- tion ; ' in many instances no phenomena have ensued, and all the results obtained are explicable by disregard of hygienic laws. In three counter-exiDeriments distilled water was inoculated. In one case more remarkable results ensued than in any case inoculated with ' rocio,' in a second the results wei-e as mild, and in a third case no results at all ensued. Finally, as the result of experi- ments, the inoculation of ' rocio ' is ineffective, and equally as negative as inoculations of black and of bilious vomit." BIBLIOGRAPHY. Carraona y Valle : LeQons sur I'etiology et la prophylaxie de la flevre jaiiue. Mexico, 1885. Fiiilay : Inoculations for Yellow Fever by Means of Contaminated Mosquitoes. American Journal of the Medical Sciences, Phila- delphia, 1891, vol. cii., p. 264. 320 IMMUNITY AND SERUM-TIIEP.APY. Fiulay : Yellow Fever. Edinburgh Medical Journal, July, October, and November, 1894. Freire : Doctrine microbienne de la tievre jaune, et ses inoculations pre- ventives. Rio de Janeiro, 1885, 630 pp. Resultats obtenus par I'inoculatiou preventive du virus attenue de la fievre jaune. Paris, 1887. ISlernberg : Report on the Etiology and Prevention of Y'ellow Fever. Washington, 1891, 265 pp., 21 plates. Report upon the Prevention of Y'ellow Fever by Inoculation. Washington, 1888. Dr. Finlay's Mosquito Inoculations. American Journal of the Medical Sciences, Philadelphia, 1891, p. 627. INDEX. Abuin, immunity from toxic ac- tion of, 53 Acquired immunity, 34-71 Alexins, 28, 46 Alkalinity of blood in immune an- imals, 26 Anthrax, 75-98 albumose, preparation of, 89 bacillus, attenuation of viru- lence, 77, 87 bacillus, chemical products of, 90,91 cured by cultures of S. pyo- genes, 243 immunity of rats, 87 inoculations, Cenkowski's method, 92, 93 inoculations, Chauveau's method, 81, 82 inoculations, Pasteur's meth- od, 80 inoculations. T o u s s a i u t ' s method, 76, 82 inoculations, priority in mak- ing, 75 inoculations, results of, 85, 91, 93 serum-therap}^ 95 spores not formed at 43^ C, 77 susceptibility to, 3 Antidiphtherin, Klebs's, 168 Anlitoxins, 24 characters of, 46 discovery of, 46 explanation of production of, 57-58 in milk of immune animals, 53, 159 mode of action of, 61 Antitoxin of diphtheria, results of treatment with, 163-168 of pneumonia, 220 of small-pox (V), 231 Antitoxin of serpent venom, 60 of tetanus, 47, 258 unit, Behring's, 157 Aronson, method of producing diphtheria immunity, 158 Attenuation of virulence, 37-39 Bacteria, pathogenic, in healthy persons, 13 role of, in nature, 12 sapropliytic, 13 Behring, method of estimating im- munizing value, 156 Black leg, See Symptomatic au- thrax Blood-serum, germicidal power of, 17, 20 antitoxic value of, 156 Buchner's explanation of immu- nity, 25 Chauveau's niethod of produc- ing immunity, 82, 85 explanation of immunity, 81 Chemiotaxis, 16 Chicken cholera, 99-110 cholera, attenuation of viru- lence, 105 cholera, augmentation of viru- lence, 105 cholera, etiology of, 104 cholera, identical witli Da- vaine's septic;emia, 103 cholera, infection by feedinu', 100 cholera, Kitt's method of yixo- ducing inmiunity, 109 cholera, protective inocula- tions in, 104 cholera, results of protective inoculations, 108 cholera, susceptible aninuds, 99 322 INDEX. Chicken cholera, Toussaint's meth- od of inoculation, 107 Cholera, 111-147 aulitoxin, in milk, 16 cultures fatal to guinea-pigs, 114 cultures, increase in virulence, 115 Ferran's protective inocula- tions, 123-130 Haffkine's protective inocula- tions, iaO-140 production of immnnitv in an- imals, 114-119 prophylaxis, 144 second attacks of, 120, 121 serum-therapy, 141-143 spirillum, attenuation of viru- lence, 116 spirillum, toxic products of, 111-114 Cobra venom, neutralized by tet- anus antitoxin, 61 Crotou-oil, immunit}^ from action of, 63 Davaine's septicaemia, 102 Defensive proteids, 23 Diphtheria, 148-168 antitoxin, Arousnn's, 158 antitoxin, Behring's, 155-159 antitoxin, Roux's, 160-163 antitoxin, results of treatment with, 163-168 bacillus, attenuation of, 150 bacillus, toxic products of, 153 bacillus, where found, 151 immunization of goats, 158 immunization of horses, 161 mortality, statistics of, 164 natural inimunit}^ of rats and mice, 151 production of immunity. 154 susceptible animals. 149 toxic action of filtered cultures, 152 Epidemics, Pasteur's explanation of, 78, 106 Erysipelas, immunity from, 243 Ferra^''s method of inoculation against cholera, 122-130 Foot-and-mouth disease, 172 Fowl cholera, iSee Chicken chol- era GERMic'iDAr. action of blood-se- rum, 21, 57 Glanders. 174-181 bacillus, toxic products of, 174-176 diairnosis of, by use of mal- leine, 178-180 production of immunity from, 177 serum-therapy, 180 susceptibility to, 3 Globulin, germicidal action of, 33 Haffkine's method of inoculation against cholera, 130-141 Hankin's method of producing im- mnnitv, 89 Hog cholera, 182-185 cholera, bacillus, toxic prod- ucts of, 184 cholera, protective inocula- tions in, 183, 184 Hog erysipelas, 186-190 erysipelas, protective inocula- tions in, 186-188 ervsipelas, serum-therapy in, 188-190 Hydrophobia, 192-206 immunity from intravenous injections, 201 immunit}', Italian method of producing. 202 immunity, Pasteur's method of producing, 194, 196 period of incubation, 195 protective inoculations in doc:s, 195 protective inoculations, inten- sive method, 198 protective inoculations, results of, 198-201 serum-therapy in, 202-205 virus, where found, 194-195 Immunity, See also Protective in- oculations acquired. 34-71 acquired, author's explanation of, in 1881, 41 acquired, Bushner's explana- tion of, 25 acquired, Metchnikoff's ex- planation of, 16-20 acquired, bv injection of fil- tered cultures, 40, 49, 293 INDEX. 323 Immunity, acquired, from anthrax infection, 80-95 acquired, from cliolera infec- tion, 114-141 acquired, from diplillieria, infection, 154-163 acquired, from glanders, 177- 180 acquired, from hydrophobia, 195-202 acquired, from septicaemia, 238-239 acquired, from pneumonia, 318-223 acquired, from small-pox, 230- 232 acquired, from streptococcus infection, 241-242 acquired, from tetanus, 256- 262 acquired, from tuberculosis, 278-283 acquired, from typhoid infec- tion, 293-295 acquired, from vegetable tox- albumins, 51-54 acquired, from venom of ser- pents, 56 natural, 3-33 natural, explanation of, 14, 31 natural, neutralized by anaes- thetics, 10 natural, neutralized by chemi- cal substances, 6 natural, neutralized by fa- tigue, 9 natural, neutralized bj^ inani- tion, 9 natural, neutralized by i)utie- factive products, 8 natural, race, explanation of, 5 Immunizing value of blood-serum, 156 Infection, predisposing causes. 10 influence of quantity of infec- tious ma-terial, 11 Influenza, 207-208 protective inoculations in rab- bits, 207 Influenza in horses, 209-210 Klebs's antidiphtherin, 168 Koch's discoverv of the tubercle bacillus, 271 mctiiod of preparing tubercu- lin, 276 Leucocytes, increase of, in infec- tious diseases, 30 Mallein, method of preparing, 175-178 use of, in diagnosis of gland- ers, 178-180 Metchnikoflf theory of immunity, 16-20 ^lilk, antitoxins in, 53 diphtheria antitoxin in, 159 tetanus antitoxin in, 261 preparation of antitoxin from, 54 NucLEiNS, chemical characters of , 30 germicidal power of, 28 Pasteur's pioneer researches, 77- 81 method of attenuating viru- lence, 37 Pathogenic bacteria in healthy in- dividuals, 13 I virulence, causes of variation in, 11 ; Phagocytosis, 14 Pleuro-pneimionia of cattle, 211- I 215 ' Pneumonia, 216-226 immunity from, 218-223 micrococcus of, 217 predisposing causes, 10 serum-therapy in, 224 j Protective inoculations. See also \ Immunity I inoculations, results of, in an- I thrax, 91-95 inoculations, results of, in I cholera, 120-141 inoculations, results of, in hy- dropiiobia, 195-202 inoculations, results of, in h( g cholera, 183 inoculations, results of, in hos erysipelas, 186-188 inoculations, results of, in in- fluenza, 207 inoculations, results of, in in- fluenza of horses, 209 inoculations, results of, in pleuro-pneumonia of cattle, 211-215 inoeidations, in pneumonia, 219-223 324 INDEX. Protective inooiilations, in small- pox, 228-2cil iiKjcuhitioiis, in streptococcus infection, 241-244 inoculations, in swiue plague, 238, 240 inoculations, in symptomatic anllirax, 24G-2.'5() inoculations, in tetanus, 258- 2G0 inoculations, in typhoid infec- tion, 29:J-295 inoculations, in tuberculosis, 278-283 inoculations, in yellow fever, Carmona's, 310 inoculations, in yellow fever, Finlay's, 311-313 inoculations, in yellow fever, Freire's, 305-309 inoculations, in yellow fever, Humboldt's, 3i3-318 Rabbit septicaemia, 237 Rabies, See Hydrophobia Rattlesnake venom, immunity from, 56 Rausch brand, 245-250 Ricin, immunity from toxic action of, 52 Rinderpest, 227 Rotlilauf, Sef. Hog erysipelas Rouget, See Hog erysipelas Roux, method of producing anti- toxin of diphtheria, 160-162 SCHWEINESEUCHE, 237-240 Second attacks in infectious dis- eases of man, 35 Serum-therapy, in anthrax, 95 therapy, in cholera, 141-143 therapy, in diphtheria, 163- 168 therapy, in glanders, 180-181 therapvt in hog erysipelas, 188-190 therapy, in hydrophobia, 202- 205 therapy, in influenza, 207 therapv, in pneumonia, 234, 225 " therapy, in small-pox, 235 therapy, in streptococcus in- fection. 242 therapy, in tetanus, 266-268 therapy, in typhoid fever, 296, 297 Serum • therapv, in tuberculosis, 287-289 Shakespeare, report on Fcri'iln's inoculations against cholera, 125- 130 Small-pox, 228-236 after vaccination, 230 antitoxin of (?), 231-234 protective inoculations, 228 second attacks of, 230 Staphylococcus infection, immun- ity from, 244 Sterilized cultures, immunitj' pro- duced by, 40 cultures, in treatment of ty- phoid fever, 297-299 Sternberg's explanation of acquired immunity, 41-45 Streptococcus infection, 241-244 infection, immunity from, 241-244 Susceptibility, race differences, 4 of voung animals, 4 Swine plague, 237-240 plague, protective inoculatious in, 238 Symptomatic anthrax, 245-250 , anthrax, preparation of " vac- cines," 246, 248 anthrax, pr(>tective inocula- tions in, 246-250 Tetanus, 251-270 antitoxin of, 48, 258-2C3 antitoxin, dose of, 261 antitoxin, in milk, 53, 261-263 antitoxin, immiuiiziug value of, 263 bacillus, cultivated in thymus bouillon, 261, 264 bacillus, toxic products of, 253-256 bacillus, where found, 252 immunit}-, 47, 256-260 immunity, inherited, 266 immunity, in horses, 265 immunity, Behring's methbd of i)roducing, 259, 260 susceptible animals, 2.")2 serum-therapy in, 266-268 toxalbumin, 254, 255 Thymus bouillon, 38. 50. 261 Toussainl's method of attenuating virulence, 37, 82 Toxins, neutralization of, in vitro, 59 INDEX. 325 Tuberculin in diagnosis of tuber- culosis, 286 in treatment of tuberculosis, 283-286 nielhods of preparing, 275-278 Tuberculocidin, Klebs's,' 277 Tuberculosis, 271-289 bacillus of, attenuated, 273 bacillus of, biological charac- ters, 272 bacillus of, discovery, 271 bacillus of, toxic products, 274 bacillus of, where found, 272 immunity from, 51, 281 results of treatment with tu- Iterculin, 283-286 serum-tlierapy in, 287-289 susceptibility to, 3 Tj'phoid bacillus, toxic products of, 292-293 bacillus, where found, 292 fever, 292-300 Tvphoid fever, serum-therapv in, 296-297 fever, treatment bv sterilized cultures, 297-299 infection, immunity fiom, 293-295 infection in lower animals, 8 infection, predisposing causes, 8 Typhotoxin, Brieger's, 292 Vaccination, discovery of, 36 necessity- for repeating, 231 Vaccinia in the cow. 229 Vibrio jMetchnikovi. inimunitv from, 115 Yellow Fever, 301-320 fever, etioloiiy not determined, 301 fever '' germ " of Freire. 802 fever, protective inoculations in, 305-319 UNIVERSITY OF CALIFORNIA LIBRARY Los Angeles This book is DUE on the last date stamped below. oJL25mS JUL 7 ^°^ APR ^ ? m \^ f^R'^^ /IPj APR2 3 '87 BiOiViEO mir88 BTOMEO. ue. 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