zu

zielenden überlegen sein sollen. Sie sind geneigt, diese an Tieren gewonnenen Resultate auch auf die Schutzimpfung des Menschen übertragen; zurzeit ist diese Impfmethode von Strong in Manila an zahlreichen Menschen angewendet worden, ohne daß sich bis jetzt eine Schädigung durch die Injektionen ergeben hat.

Immerhin ist die Zahl der Geimpften noch zu gering, um ein Urteil darüber abgeben zu können, ob beim Menschen die KolleStrongsche Methode dem bisher üblichen Verfahren der Schutzimpfung mit abgetöteten Bakterien tatsächlich vorzuziehen ist. Auch wird man vorläufig eines gewissen Bedenkens sich nicht entschlagen können, daß diese Impfungen mit lebenden, wenn auch noch so sehr abgeschwächten Pesterregern doch eine gewisse Gefahr für den Geimpften involvieren. (Wenn auch nur einer von Tausenden infolge der Einverleibung des lebenden Vaccins stürbe, so würde dies doch ein Argument sein, welches dieser Impfmethode gegenüber die größte Zurückhaltung zur Pflicht machte.)

Ueber das Terni-Bandische Impfverfahren, bei welchem sterilisiertes Bauchhöhlenexsudat von Meerschweinchen, die einer intraperitonealen Pestinfektion erlegen sind, injiziert wird, sind wir zurzeit noch ungenügend orientiert. Es ist unmöglich zu sagen, ob dieser Impfmethode ein Vorzug innewohnt, der die Schwierigkeit der Impfstoffgewinnung wettmacht.

Ich glaube gezeigt zu haben, daß auf dem Gebiete der Schutzimpfung des Menschen gegen die verheerendsten Seuchen, Cholera, Pest, Typhus, noch keineswegs das Ideal erreicht ist. Doch eröffnen sich mannigfache, viel versprechende Wege und wir dürfen hoffen, daß die unablässige Arbeit zahlreicher Forscher aller Nationen uns in absehbarer Zeit dem Ziele völlig sicherer und dabei gefahrloser Impfmethoden entgegenführen wird.

V, 5

Preventive inoculation against typhoid fever, plague, cholera.

Protective inoculation against plague.

By

Prof. Dr. Strong (Manila).

Before immediately opening the discussion of the subject to which I have been assigned, I wish to express my gratitude to the Committee on organization of the Congress for the honour which they have conferred upon me by inviting me to appear before you upon this occasion and at the same tome to remind you that before an audience of this nature it is naturally with considerable diffidence I approach the consideration of the subject of protective inoculation against plague.

The important and extensive investigations upon plague immunity which have been carried on in earlier years by Yersin and Kitasato, Gaffky, Pfeiffer, Sticker and Dieudonné, Albrecht and Ghon, Haffkine, Bannermann, Klein and others are so well known that they need only be mentioned: the more recent ones performed by Kolle and his assistants during the past few years in the Königliches Institut für Infektionskrankheiten of this city have attracted such wide attention and have been so comprehensive that for this reason and because so many of you are so much more familiar with this topic in general, than I am, I fell that I should not be justified in asking your attention to my remarks, even for a few moments, were it not for the fact that some of my experiments would appear to elucidate somewhat further several points of practical importance in connection with this question of protective inoculation.

Since Manila has, in former years, suffered from Bubonic plague and is in constant danger from another invasion of this malady, brought about by the introduction of the infection, either from Hong Kong or certain of the Japanese and Indian Ports, it is necessary for us to devote at least a part of our attention to the practical problems in combating this disease and in my Laboratory it is particularly from this standpoint that this subject has been investigated. During the past year and a half 1 have undertaken experiments with the object of throwing further light upon certain practical problems in relation to pest immunity and particularly with the idea of determining the most efficacious method of protective inoculation against plague.

The subject of immunization against pest is not only of general scientific interest, but to many tropical and sub-tropical countries is of great practical importance. One need only recall the mortality in India of nearly a million deaths from this disease during the year 1905 and of over one million during the first six months of the present year, to be impressed with the significance of the problem.

It is true that the subject of inoculation against plague has received considerable attention during the past few years and that prophylactics have been recommended by several authors, but while it is admitted that by their use a certain degree of pest immunity can be produced and demonstrated in a number of the more insusceptible animals and occasionally even in those very susceptible to this infection, nevertheless it has sometimes seemed questionable whether we were able by their inoculation to obtain in man an immunity of such a degree as to be protective against the natural and usual methods of infection of the malady.

In the latter part of the year 1903, when the Board of Health of Manila was practising among the Chinese of that Citv, protective inoculation against plague by the injection of the killed cultures of the pest bacillus, the method at that time carried on in Japan and consisting of the injection of one oese of a 24 hour slant agar culture of Bacillus pestis suspended and killed in saline solution, I decided to investigate whether any immune substances became developed in the serum of the inoculated individuals. I therefore studied the agglutinative and bactericidal reactions of the blood serum of 12 cases, six of whom had been inoculated two weeks and six three weeks previously with one oese of the killed pest culture. The agglutinative reactions were performed by the macroscopic method, and the bactericidal reactions according to the one suggested by Neisser and Wechsberg. No traces of agglutinins or of specific bacteriolysins could be demonstrated in the sera of any of the individuals. Obviously, these experiments in themselves were not considered to be conclusive evidence of the fact that no immunity was conferred upon the inoculated, since it was already recognised at this time that these anti-bodies were frequently and indeed, usually, not encountered even in the blood sera of individuals who had recovered from an attack of plague and were immune to this disease. Therefore, experiments, in animals were resorted to in order that more information on this subject might be obtained. Guinea-pigs were inoculated subcutaneously each with the same dose that was being employed in the general human inoculations in that City. After two weeks the immunity of these animals was tested in the following manner. One oese of a virulent pest organism was suspended in one cubic centimeter of saline solution and 5 oeses of this suspension rubbed over a freshly shaved area on the abdomen of the guinea-pig. All of the animals succumbed to acute pest infection, demonstrating conclusively that an immunity of appreciable degree had not been produced. A short time after the important paper of Kolle and Otto was published in which the unfavourable results from the immunization of guinea-pigs with large doses of killed agar cultures of the pest bacillus or with Haffkine's prophylactic were reported. It therefore seemed to me, at that time, more advisable to experiment further with other methods of immunization against plague, before insisting upon the use of larger amounts of more virulent killed pest cultures in the human inoculations being pursued in Manila. Further results obtained by Kolle and his associates in Berlin and the animal experiments made in my Laboratory having demonstrated that in very susceptible animals at least, satisfactory immunization could only be exceptionally produced by the methods of human inoculation then in vogue, I recommended at that time to the Commissioner of Health in Manila a suspension of the human inoculations with killed cultures of the pest bacillus.

A comparative study of the efficacy of the different methods of protective inoculation against plague was then undertaken and their value investigated not only in guinea-pigs but particularly in the lower apes.

The methods of immunization, especially studied, consisted of 1. the inoculation of killed bouillon and killed agar cultures; 2. of living attenuated cultures (that is vaccination); 3. of filtered cultures and extracts (free receptors of the organism); 4. of artificial and 5. of natural plague aggressin and 6. of inoculations according to Klein's method. I may say in passing that in this Paper I employ the term „vaccination" as implying exclusively an inoculation with the living attenuated organism.

The results of this comparative study have been recorded in detail in various papers during the past and present year most of which have been published in the Philippine Journal of Science. However, a comparison of the value of the different methods may also be made by a glance at the wall chart (which require no explanation).

These experiments have demonstrated conclusively and beyond any doubt the great value of vaccination against plague infection in animals and its evident superiority to the other methods of immunization.

Next in value to vaccination as a means of immunization against pest appear to be prophylactic inoculations of natural aggressin. Inoculations with artificial plague aggressin did not in my experiments prove to be nearly as efficient as those with natural aggressin. However, as I have pointed out previously, there was apparently no difference in the quality of the immunity obtained with the natural aggressin from that produced by the artificial product and my subsequent experiments as did my earlier ones, have only further confirmed the views of Wassermann and Citron that the agressins must be considered to be hypothetical substances and that, so far as their immunizing value is concerned, in these exsudates we have to do mainly with the substances extracted from the bacilli themselves. Evidently, in the case of the plague bacillus, the receptors of the organism in the socalled agressin exsudates, of animals become liberated in a more efficient manner for immunization and probably exist in a less altered condition than they do in the aqueous suspensions of the bacilli obtained in vitro by artificial means.

However, the method of inoculation with natural plague aggressin is not likely to come into general use because of the great difficulties encountered in the preparation of the prophylactic. Moreover, in my experiments I have not obtained the satisfactory results with it which Hueppe and Kikuchi evidently anticipated.

After having concluded from the experiments mentioned above that true plague vaccination (that is inoculation with living attenuated cultures) produced the highest immunity inoculations were made in human beings with a living culture designated as „Pest Avirulent". This culture was obtained by me from Professor Kolle who originally received it from Dr. Maassen. In order to ascertain if any evidences of immunity could be demonstrated in the inoculated, their blood was tested for the presence of agglutinins anti-infectious substances and opsonins. This led me to investigate in detail whether and if so to what extent these same anti-bodies existed in the blood serum of animals which had been immunized against plague infection. From a large number of experiments I have concluded that the agglutinins are formed slowly and only in very small amounts in animals which are being immunized against pest infection and that they only occur in demonstrable quantities in those which have been very highly immunized. At most, only very minute traces of these substances are encountered after single inoculations of either the killed or the living organisms, no matter how large the dose which is employed. I am convinced that in my earlier experiments with plague agglutination I sometimes mistook pseudo-agglutinations of the pest bacillus for true ones, and from a study of the literature it seems to me very likely that other observers have also erred in this respect. A study which I have made of the blood sera of guinea-pigs and moneys, which have been vaccinated against pest infection and which have later shown themselves to be immune to lethal and multiple lethal doses of the pest bacillus, has demonstrated that practically no traces of agglutinins exist in such sera.

Only small quantities of agglutinins could be detected in the examination of several pest immune sera which were prepared in horses and which were known to possess, in two instances at least, considerable protective (that is anti-infectious) power.

A study of the agglutinating properties in the blood of a number of persons who had been vaccinated against plague as well as of several cases who had suffered with the disease and recovered was also undertaken. In the majority of the instances no traces of agglutinins could definitely be demonstrated.

The method employed in investigating the presence of antiinfectious bodies in the serum was as follows: A rat was inoculated intraperitoneally or subcutaneously with the serum to be tested while at the same time the animal was infected by thrusting beneath the skin near the tail a syringe needle which had been dipped in a suspension of a virulent pest-organism. Numerous control animals were inoculated in all the series.

By extensive experiments of this nature it was demonstrated that the anti-infectious substances also become developed very slowly and in very small quantities in animals immunized against pest. While rabbits which have been given a single, small, intravenous inoculation