PAPERS READ.

DISCOVERY OF THE PRODUCTION OF IMMUNITY FROM CONTAGIOUS DISEASES BY CHEMICAL SUBSTANCES FORMED DURING BACTERIAL MULTIPLICATION.

By Dr. D. E. SALMON, U. S. Agricultural Department, Washington. Ar the meeting of this Association held in Buffalo in August, 1886, I had the honor to read before the Biological section a paper entitled, "Immunity from Contagious Diseases produced by products of Bacterial Multiplication," and a second paper on "The Theory of Immunity from Contagious Diseases." In the first of these papers I gave a detailed statement of nine different experiments in which forty-five pigeons were used. The results of these experiments demonstrated incontestably that these birds, which were susceptible to the dose of icc. of hog cholera virus, might be made immune from this dose by hypodermic injections of sterilized culture liquid in which the germ of this disease had been previously allowed to multiply. Nearly a year and a half after the reading of this paper (Dec. 25, 1887), MM. Roux and Chamberland, of Pasteur's Laboratory, published a paper in the Annales De L'Institut Pasteur in which they gave experiments to show that guinea pigs may be made immune from the effects of the vibrion septique by inoculation with sterilized culture liquids in which this germ had been allowed to multiply previous to sterilization. In a footnote to this paper they say, "At the moment of going to press we read in the Annual Report of the United States Department of Agriculture, that M. Salmon has succeeded in giving immunity to pigeons from hog cholera by injecting into these animals sterilized cultures of the microbe of this disease."

In an article in the Annales De L'Institut Pasteur of February 25, 1888, Roux publishes another article showing that guinea pigs may be made immune against the virus of symptomatic anthrax by injections of cultures in which had been grown the bacterium chauvai, which cultures had afterwards been sterilized by heat. In the same number of this periodical MM. Chantemesse and Widal publish an article on "Immunity against the Virus of Typhoid Fever by soluble substances." In this article they make the following statement: "In 1886 Salmon rendered pigeons refractory to hog cholera by inoculating with sterilized cultures of the microbe of this disease The experiment was of little weight [était peu concluante] because the pigeon is endowed with a feeble receptivity for this virus and because the process of vaccination applied to pigs and other mammals completely failed. There was wanting an incontestable experiment which showed immunity given to an animal species very sensitive to the virus by means of soluble

substances secreted by this virus. This experiment and this demonstration are found in a memoir that MM. Roux and Chamberland published in the month of December in the Annals De L' Institut Pasteur on the 'Immunity against Septicæmia.' This work inaugurates a new era in bacteriology." Hueppe in an article in the Fortschritte der Medecin, for April 15, 1888, takes up the history of the investigations of this subject properly giving credit to my experiments, the first of which were reported conjointly with Dr. Smith before the Biological Society of Washington in February, 1886. He shows that the objection of Chantemesse and Widal is without foundation, because of the check animals used in our experiments.

There is still a determined effort being made, however, by the assistants of Pasteur to secure the credit of this discovery for the Pasteur laboratory. In a review of my investigations of hog cholera written by Duclaux and published in the Annales De L' Institut Pasteur for July 25, 1888, the subject is again taken up and a labored effort made to show that my experiments were not conclusive. M. Duclaux begins his review by saying: "The works of M. Salmon on hog cholera are scarcely known in France and appear to be known little better to the European public. There are two reasons for this. The first is, that they have been published in an official report not found in commerce, and the copies which reach Europe generally go to be buried in the tomb of the ministerial archives,or in the grand mausoleums hostile to visitors that we name, in France, public libraries. The other reason, more personal to the author, is that he proceeds too much by that method of exposition which consists in pouring on the head of the reader the entire contents of his notes of researches, leaving him to make his choice among the materials which thus reach him without order and without measure. . . As I said in the beginning, they are not known in Europe where they scarcely figure in the journals until after the tardy communication which was made in 1887 by MM. Salmon and Smith to the Washington congress. But had they been known sooner, they would not have been sufficient to rally the then hesitating opinion on the subject of vaccination by soluble substances. They exposed the flank too much to very legitimate objections."

Before considering the objection raised by Duclaux to the experiments themselves, I desire to call the attention of the association to certain publications in which these experiments were referred to at considerable length prior to the meeting of the International Medical Congress at Washington. The first communication was read before the Biological Society of Washington, Feb. 20, 1886, and printed in volume II of the Proceedings. This paper was printed in full in the American Veterinary Review for May, 1886, and it was referred to at considerable length in an editorial printed in the New York Medical Journal of March 6, 1886. This paper contained our first experiments with sterilized culture liquids and in it was given the demonstration of the principle, that immunity may be produced by the injection of soluble substances which result from the growth of pathogenic bacteria. The paper was confined to one topic, was concisely written and occupied less than five pages, and consequently is hardly

open to the criticism of M. Duclaux. The paper read before the American Association for the Advancement of Science in August, 1886, was printed in full in the Proceedings for that year, p. 258, and was also printed in the Medical News of September 18, 1886. It appears to me therefore that if the scientists connected with M. Pasteur's laboratory were ignorant of these investigations until after the meeting of the International Medical Congress in September, 1887, it is not because the literature was inaccessible.

MM. Chantemesse and Widal take exceptions to my experiments because pigeons, which were the animals used, were but slightly susceptible to the virus, and because this process of vaccination applied to hogs and other mammals had completely failed. It is somewhat amusing to find in the same number of the Annales a communication from M. Roux, of M. Pasteur's laboratory, claiming to demonstrate that immunity can be produced from Charbon Symptomatique with soluble substances and in which the investigation was made by experiments with animals even more insusceptible to this virus than were pigeons to the virus of hog cholera. Roux used guinea pigs, and he tells us that "cattle and sheep are the animals that most easily take the Charbon Bactérien the guinea pig shows more resistance to this disease; it does not always die as the result of inoculation made with the pulp of the charbonous tumor. MM. Arloing, Cornevin and Thomas have made known a mode of inoculation which always produces death to guinea pigs. To obtain this result it is sufficient to mix the virus with a solution containing twenty per cent of lactic acid. With this means of proof there need be no fear of a failure of the inoculations or of seeing the control guinea pigs resist them."

It is a rather peculiar idea to advance that we must receive without question experiments made in Pasteur's laboratory with animals so insusceptible that a foreign substance must be added to the virus in order to produce positive results, while exactly similar experiments must be rejected, when made in this country with virus, to which it is not necessary to add any foreign substance, and which, under the conditions mentioned, never failed to produce positive results. This objection was apparently disposed of by Hueppe, however, when he called attention to the control experiments and the positive results of these inoculations.

The question is, however, apparently deemed of too much importance to Pasteur's laboratory to be allowed to rest here. In Duclaux' review a new objection is relied upon to show that my experiments were valueless. It is that the liquids were sterilized at a temperature too near to the limit at which the microbe is destroyed. Duclaux claims that testing this liquid by adding a few drops to a fresh culture tube, in order to learn whether there are any living organisms present, proves nothing. "It was known," he goes on to say, "and it is still better known now, that microbes which are present in a culture are not identical, and that they have different powers of resistance to external agents. These powers of resistance are not identical, although approaching each other closely, and the more closely, the more homogeneous the culture. Nothing proved then and nothing proves yet, that all of the microbes were killed at this temperature limit of 58°-60°."

These objections were anticipated when our experiments were planned, and anyone who will go carefully into the details of these experiments will see that the objections do not hold good. It is true that the temperature of 58°-60°, at which we sterilized our culture liquids, is rather near to the limit which is necessary to destroy all life in these cultures. While admitting that there may be different degrees of resistance with this germ, however, we demonstrated that 58° maintained for ten minutes was sufficient to destroy those germs with the greatest powers of resistance, because none were left capable of multiplying after they had been so treated. In our experiments on immunity, we do not limit the cultures to a temperature of 58° for ten minutes; on the contrary, we increased the temperature one of two degrees and we maintained this increased temperature for two hours in other words for twelve times as long as had been found necessary to destroy all living germs. It will also be seen by reference to the details of our experiments that, in some of these, the culture liquids employed were concentrated over a waterbath. In these experiments the culture liquids were heated to a temperature of nearly 100° for from one to two hours. This is far beyond the point where there could be any doubt of the germs being completely destroyed. While some of the experiments in which this evaporated virus was used were successful, we recognized the fact, apparently confirmed by the French investigations, that such a temperature weakened the power of the liquid for the production of immunity. We consider, therefore, that this objection of Roux is absolutely untenable and that there can be no question that the virus was completely sterilized in our experiments.

As another reason for his belief, Roux cites the small quantity of sterilized culture which was sufficient to produce immunity. "Thus," he says, "in the first of these experiments published in the Report of 1885, a dose of cc. of vaccinal culture sufficed to confer almost complete immunity on a pigeon. This dose differs greatly from that which is necessary to confer immunity in other diseases." Roux evidently errs in this statement. By referring to the Report of 1885 it will be found that one pigeon received cc., and when inoculated with strong virus died in forty-eight hours, i. e., this bird had no immunity whatever. Another one received 1.5 cc., and when inoculated with active virus was so affected that it lost the use of its legs although it survived the inoculation. In this case there was evidently a partial immunity. All of the other birds referred to in that Report received over 3 cc. In our subsequent experiments we found that 2 cc. would generally confer immunity on pigeons, although in many experiments we used 3 to 4 cc.

When we turn to the experiments made in Pasteur's laboratory, we find that this quantity is not so different from what was used there as we would be led to believe from the remarks quoted. For instance, while in the experiments of Roux and Chamberland, for producing immunity in guinea pigs against the Vibrion septique, they used 120 cc. of culture liquid which had been heated to 105°-110°. They succeeded in producing equal immunity with doses of only 1 cc. of the serum from the muscles and cellular tissue, which was sterilized by filtration instead of by heat. It would appear