over sulphuric acid in vacuo, giving white translucent crystalline plates. After drying it was still soluble in water, though dissolving with more difficulty. The water solution gave with platinum chloride an almost soluble precipitate, appearing under the microscope as needle-like crystals. The composition of this platinum salt shows it to be a substance allied in composition to peptone. As to the exact nature of this latter substance, whether it is a true proteid or belongs to the class of ferments, remains to be determined by subsequent study and investigation. Brieger and Fraenkel (Ber. Klin. Woch., 1890, No. 11), who have extracted a similar substance from culture liquids of the diphtheria, tetanus, and cholera germs, and Baginsky and Stadthagen (Ber. Klin. Woch., 1890, No. 13), who obtained an allied body from cultures of the cholera-infantum germ, hold that the substances are proteids. Roux and Yersin (Annales de l'Institut, Pasteur, 1890, p. 385), on the contrary, hold that the substances obtained by the precipitation with alcohol are ferments. Hankin (British Medical Journal, July 12, 1890, p. 65), who has also isolated a substance from cultures of anthrax possessing albuminoid properties, holds the same view as Brieger and Fraenkel, that the body in question belongs to the class of proteids. In so far as our work upon the hog-cholera culture liquids goes, we are inclined to the opinion that we have to deal with albumoses, which can be heated in presence of acids to 70° without decomposition. I am preparing now a considerable quantity of this albumose and hope to be able in a short time to have something more definite as to its exact nature. To be sure that the substance one is dealing with is absolutely pure is very difficult when it is a body of this nature, and only extended experiments can be regarded as conclusive. As to the nature of the new ptomaine which has been isolated, we will not go into a discussion of its exact chemical composition until it is more definitely determined. In order, however, to distinguish the active principles formed by the hog-cholera germ, I have named the ptomaines as a class sucholotoxins, and the new base sucholotoxin (from the Greek Los, a hog, Xoλépa, cholera, from Xoir, bile, and Totzó, poison). To the proteid body I have given the name sucholoalbumin. These names will be used in referring to these bodies in the future. Some experiments were made later, but may be inserted here, in regard to substituting some other material for peptonized beef infusion in furnishing nourishing media for the artificial cultivation of the germ. Potato broth, pea broth, and plain beef infusion have been used. In all of these the hog-cholera germ grows very vigorously, forming the ptomaines and albumoses, but not in so large a quantity as in the peptonized beef infusion. Now, in regard to the toxic effect of the sucholotoxin and sucholoalbumin, active poisons for guinea pigs, in small doses, they are not. In large doses, corresponding to from 6 to 15 cubic centimeters of the culture liquid, death is produced in guinea pigs in from six to twenty-four hours. A small subcutaneous injection causes the animal to appear stupid and uncomfortable for a short time, fifteen minutes, produces a slight rise in temperature, necrosis of tissue, and ulceration at the point of injection. It may be added here that in making these and all the following experiments special precautions were taken to prove that the mate rial used was entirely free from germs. from the substances used for injection. Cultures were always made The autopsy of a case resulting from poisoning with the ptomaines may be inserted here: Liver, pale and fatty; subcutaneous tissue over abdomen necrosed, and infiltrated muscle soft and friable. Other organs apparently normal. The next point to be decided was: Can immunity be produced from hog cholera by previously treating the animals with these substances, isolated from the culture liquids? The results are recorded in the following experiments, which are very conclusive. For the laboratory experiments guinea pigs were used as being convenient to handle and susceptible to hog cholera. They have proved very satisfactory. The first of our experiments that we will record were made with sucholotoxin. Experiment I.-Two guinea pigs, each weighing about threefourths of a pound, were treated with a solution of about 0.05 gram of sucholotoxin hydrochloride each. The solution was introduced under the skin of the inner side of the left thigh. Immediately after the operation the animals appeared uncomfortable, but were not made ill. For a few days there was a rise in temperature and also a slight swelling at the point of inoculation, which, however, disappeared in about five days, and the animals were then well. Two more guinea pigs were now selected as checks, approximately of the same size and weight as those which had been treated, and the four animals were then inoculated with 0.1 cubic centimeter of hog cholera virus each (0.1 cubic centimeter beef infusion peptone culture one day old, plus 0.2 of sterile, normal salt solution). This is the dose which previous experiments made in the Bureau had shown to be the proper quantity to kill a guinea pig in from eight to ten days. The inoculations with the virus were also made subcutaneously in the thigh. The checks died in eight and nine days. Of the animals which had been first treated with the substance mentioned, and afterwards inoculated, one died two days after the last check. The other guinea pig of this set was quite ill for ten days, with a large swelling at the point of inoculation. This finally opened and healed, and the animal was quite well within three weeks after the inoculation, and has continued so to date-five months. Experiment II.-The next series of experiments were made with sucholoalbumin from beef infusion peptone culture media. Two guinea pigs were again selected and treated with about 0.008 gram each of sucholoalbumin. There was a slight rise of temperature in the animals and the formation of a small, hard lump at the point of injection. This disappeared by the eighth day and the animals were quite well. Two more guinea pigs were now taken as checks, and all four animals were inoculated with 0.10 cubic centimeter of hog cholera culture. The checks died within seven days. The post-mortem appearances were practically the same as those noted in the first series.. The two guinea pigs which had been treated with the sucholoalbumin died ten days after the checks. This indicates considerable resistance to the disease. Several other experiments were made by treating guinea pigs with the albumin in varying quantities, all showing resistance, and subsequently immunity. Experiment III.-Three guinea pigs were treated with sucholoalbumin, 0.1 gram being given to each, subcutaneously in the thigh. The albumin for two of the animals was derived from cultures containing blood serum, the albumose given to the third was from ordinary beef infusion peptone culture. Ugly ulcers formed at the point of inoculation, which healed, however, in from ten to fourteen days, and the animals, with the exception of a slight rise of temperature, were well. Two checks were again selected and the five animals were inoculated with 0.10 cubic centimeter hog cholera virus. The checks died, respectively, in eight and ten days from hog cholera. The animals which had received the preventive treatment were slightly ill for a few days with swelling at the point of inoculation, which finally opened and then healed nicely, and within a week the guinea pigs were well. Three weeks after the inoculation one of these animals was chloroformed and examined post-mortem. Not the slightest scar could be discovered, all the organs appeared perfectly normal, and no germs were found. Experiment IV.-Four guinea pigs were treated, two with a mixture of sucholotoxins, two with sucholotoxin and albumin. The injections were made as before, subcutaneously in the thighs, and at intervals extending over a period of four weeks. The sore caused by each injection was allowed to heal before the next one was made. After the animals had recovered from the last treatment two checks were selected, and the six were each inoculated with one tenth cubic centimeter hog cholera virus. The checks died, one in eight and the other in ten days, the post-mortem examination showing characteristic hog cholera lesions. The animals having the preventive treatment were ill about four days, those that received only the sucholotoxins being more dull than the others. There was also slight swelling at the point of inoculation with the germ, which subsided in ten days, after which the animals were perfectly well, and have remained so four months. Experiment V.-Six guinea pigs were inoculated for this experiment, two with solution of the sucholotoxin and four with a solution of the mixed sucholotoxins. The sucholotoxin solution produced only slight local lesions, while the mixed toxins caused ulceration at the point of injection which did not heal for two weeks. The animals having by this time recovered, the test experiment with hog cholera virus was tried. Four of the animals mentioned above were takentwo from each set-and also two checks, and the six were inoculated. The checks died in eight and nine days, the autopsies showing the characteristic conditions of death from hog cholera. Those that had the preventive treatment were ill and dull for from four to six days after the inoculation. At the point of inoculation there was also some swelling and infiltration, very slight, however, compared with the similar swelling on the checks. In the treated animals the swelling sloughed and healed, and within ten days after the inoculation they were perfectly well. To test the resistance of the animals that had been treated by this method to ordinary exposure the following experiments were conducted. Experiment VI.-Two guinea pigs that had received the preventive treatment, two blanks-i. e., animals that had received no treatment-and two check animals that were inoculated with hog cholera virus were placed in one large cage. The checks became ill and died in eight or nine days from hog cholera. During this time the cage was cleaned only three times, so as to give full and free oppor tunity for contagion. One week after the checks had died one of the blanks became ill, and died within ten days. The autopsy showed hog cholera lesions. The second blank became ill a few days after the first blank succumbed, and died within thirty days. The animals which had the preventive treatment are now and have been quite well, though continually exposed for five weeks to every opportunity for contagion. These experiments have answered conclusively the first five propositions named in the beginning of this report, and brings us to the sixth. Can these substances be replaced by one of allied composition and character that we already know and can prepare synthetically in the laboratory? The experiments also give an affirmative answer to this problem. If the ptomaines when introduced into the system produce certain changes, or induce certain powers of resistance on the part of the animal to subsequent doses of the poison, then it is possible that not only this one particular alkaloid but several, belonging to the same class and of approximately the same chemical composition, should produce similar effects when introduced into the system, as the true ptomaine extracted from the culture liquids, and subsequently immunity should result, when the animal should be exposed to the virus of hog cholera. I thought of a substance which could be prepared without difficulty, and which I will refer to as pure chemical. Some of it was prepared and the solution used for injection. The injections and treatment were conducted in the same manner as already recorded for the other guinea pigs, three animals being used for this experiment. Experiment VII.-There was a slight rise in temperature of the animals and swelling and soreness at the point of injection. After this had healed these animals and two checks were inoculated with one tenth cubic centimeter of hog cholera culture. The checks died in eight and nine days. The animals which had been previously treated became ill, two dying five and six days after the checks. The third entirely recovered. Experiment VIII.—One guinea pig was treated with a solution of the chemical in the same way as the previous experiment, except that a somewhat larger dose was given. Two pigs were again taken for checks, and the three inoculated with 0.1 cubic centimeter hog cholera culture. The checks died in six and seven days, respectively, of hog cholera; the treated animal recovered entirely. In the treated animal there was a slight swelling at point of inoculation with the germ, but this gradually decreased, finally opened, sloughed, and healed within a few days after the death of the checks. Experiment IX.-Four guinea pigs were treated with a solution of the chemical substance. This modification of the injections was adopted, i. e., very small quantities were used at a time and the dose repeated every day. The local irritation was in this way much diminished and what soreness was produced healed more rapidly. Two checks were taken and the six animals inoculated with 0.1 cubic centimeter of hog cholera culture. The checks died of hog cholera in eight days, one vaccinated pig in thirteen days, the others recovered. Experiments were also made in producing immunity with the ptomaines obtained from the potato, pea, and simple beef broth cultures, which resulted successfully. Two of the guinea pigs which had recovered from experiment IV, and two that had recovered from experiment V. were now reinoculated with double the dose of hog cholera virus used in the first test. Checks were taken and given one half dose in quantity of the virus. These died in eight and nine days. The other pigs were a little stupid for a day or so, but at no time ill, and have since remained perfectly well. One pig from experiment V, and one from experiment III, were chloroformed four or five weeks after their recovery, and an autopsy made. All the organs appeared perfectly normal, not even a scar being left at the point of injection, and the immunity produced was therefore perfect. Our experiments had now proved that the chemical principles produced by the germ could be isolated; that their injection into guinea pigs rendered the animals secure against an attack of hog cholera, and that we have at hand a compound fairly easily obtained which will give the same results in securing immunity. EXPERIMENTS UPON HOGS. The next question was: Will these same materials produce immunity in hogs, and can the production of immunity by this method be made practical? The experiments were carried on at the Animal Experiment Station of the Bureau. The injections were made by Dr. Kilborne, who recorded the notes upon the condition of the animals. Necessarily the hogs were not as easy to handle as the guinea pigs, and the first experiment, which is the only one complete at this time, is not conclusive; but considering the time which must elapse before a question of this sort can be positively decided we regard the ultimate practical solution of the problem only as a question of detail, which a few more experiments will enable us to decide. In order to test the value of this ptomaine, which had proved so satisfactory for guinea pigs, and also of the synthetically prepared chemical compound upon hogs, the following experiment was conducted: Nine pigs, black Essex grade, aged three months, were selected, four of them being placed in one pen and five in another. Pig No. 374, aged three months, weight 60 pounds, treated on July 26 with solution of the ptomaine, 18 cubic centimeters of solution were used, the injection being made subcutaneously at three points. On July 30 there was a large swelling at seat of injection. By August 8 this had sufficiently healed to permit of injecting more of the solution of the ptomaine. The dose was repeated on August 16. August 20 there was swelling (lumps the size of a hen's egg), at the points of injection. These sores had healed by sloughing, and on September 9 the animal was inoculated in the femoral vein with 2 cubic centimeters of beef infusion peptone hog cholera culture, one day old. Pig No. 375, aged three months (weight 60 pounds), was treated in the same way as pig No. 374, with solution of the ptomaines, and showed the same soreness and symptoms. On September 9 inoculated with 2 cubic centimeters of hog cholera beef infusion peptone culture one day old. Pig No. 376, aged three months (weight 50 pounds), treated with ptomaines as other two and inoculated September 9 with 2 cubic centimeters beef infusion peptone hog cholera culture. Pig No. 377, aged three months (weight 50 pounds), treated on same dates as the above with a solution of the synthetical compound and inoculated September 9 with 2 cubic centimeters beef infusion peptone hog cholera culture one day old. Pig No. 378, aged three months (weight 45 pounds), treated in same way as pig No. 377, and inoculated September 9 with 2 cubic contimeters beef infusion peptone hog cholera culture. Pig No. 379, aged three months (weight 60 pounds); Pig No. 381, aged three months (weight 50 pounds); Pig No. 382, aged three months (weight 50 pounds), were all inoculated in the vein on September 9 with 2 cubic centimeters beef infusion peptone hog cholera culture one day old. These served as checks to Nos. 374 to 378, inclusive. |