Abbildungen der Seite
PDF
EPUB

INVESTIGATIONS IN SWINE PLAGUE.

During the past year the investigations concerning this disease were carried on without intermission. The methods adopted were those used by the most advanced investigators as well as those which suggested themselves to us directly and grew out of the necessities of the case. At least 20 animals were carefully examined in the earlier part of the year. In nearly all instances the disease had been communicated from the sick to the healthy by contagion, either at the experimental station of the Bureau or in neighboring herds. Very few showed any disease of the lungs. In fact, the disease manifested itself chiefly by extensive ulcerations of the cæcum and colon (Plate I). The animals lingered usually from one to three weeks after the first appearance of the disease and in nearly every case were killed in the last stages to prevent any post-mortem changes, as the animals, when left to themselves, usually die early in the night and decomposition sets in very rapidly during the spring and summer months in this climate.

It was our intention to study carefully the microbes found in the various exudates of the serous cavities, especially that of the peritoneal cavity, which has been considered very virulent by former observers. A simple and safe mode of collecting these exudates will be described in detail further on.

In cases of advanced disease, characterized by extensive ulcerations of the large intestine, it was found, on microscopic examination of lymphatic glands and other organs invested by the peritoneum, that the latter membrane was covered with a layer of lymph in which were imbedded various kinds of bacteria, micrococci of different sizes, slender as well as thick bacilli. Cover glasses brought in contact with the peritoneum likewise contained several forms. Finally the persistent impurity of the contents of vacuum tubes as well as cultures made directly from the serous effusions at the post mortem examination, forced us to conclude that we must not look for any pure culures from this source. The inference was that microbes gained access to the closed cavity through lesions caused by the extensive ulcerations of the large intestine, which always accompanied these cases, and that they were not there destroyed, either because the system had been so debilitated or the microbes were capable of a parasitic existence.

The peritoneal exudate had been pronounced virulent by Klein, who thence obtained the bacillus, claimed by him to be the cause of swine plague. We determined to isolate and study the different bacteria which we should meet with in this exudate by culture and inoculation. If the lesions in the intestine were due to a local multiplication of the specific microbe in the mucous membrane, it seemed natural to suppose that it would be very abundant in the contents of the intestinal canal, and would find its way with the other bacteria into the peritoneal cavity, and that having become adapted to the struggle with animal tissues it might even outgrow the other forms in this situation. At the same time the possibility must not be set aside that septic bacteria might gain entrance in the same way, and by a rapid invasion of the blood or lymphatic channels, or both, materially change the clinical aspects of the disease and give rise to various apparently inexplicable phenomena and sequela. In two cases a bacillus was found in the peritoneal cavity made up of long, jointed filaments, and probably identical with the bacillus of malignant cedema (ribrion septique), the spores of which, according to Koch, are abundant in the soil. These filaments

were found in great abundance on cover glasses upon which a delicate film of peritoneal exudate had been dried. In another pig, which died in the night during a heavy frost (November 22, 1884), and which was examined early next morning, a cover glass touched to the peritoneal surface of the liver was found crowded with a bacillus in long jointed filaments, no doubt identical with the preceding. The appearance of the abdominal cavity in this case, as indicated in the notes of the postmortem, are briefly as follows: Abdomen on opening emits a faint odor not observed in previous cases. Ulcers of the mucous membrane of the large intestine plainly visible through the peritoneum; peritoneum dry, no serum in the cavity, no evidence of peritonitis. Liver of a pale reddish color, on section, bloodless; cæcum and colon studded with ulcers, some covered with a projecting black, necrotic mass. Sections of the liver hardened in alcohol were found to contain three different forms of microbes, the bacillus found on the surface of the liver, the individual filaments of which were very long; a small, slender bacillus and a micrococcus; these were present in equal numbers. The cover glass with which the peritoneal surface of the large intestine had been touched in close proximity to the ulcers, revealed not a single microbe among the numerous epithelial cells which had come away. Thus the invasion of the bacillus was no doubt by way of the bile ducts in this case. The presence of such large numbers of microbes in the liver tissue can hardly be accounted for by a post mortem growth in this instance. In blood collected from the heart and examined unstained, this same bacillus was found, the elements pale and almost disintegrated.

In sections made of one of the ulcers, the mucous membrane was found entirely thrown off, as well as a portion of the submucous connective tissue. The muscular layer was replaced by an inflammatory infiltration of cells and enormously thickened, so that the line of fatty tissue in the submucosa was pushed forward into the lumen of the tube and formed two sides of a cone (the apex of which had sloughed away) between the mass of infiltrated cells. Various kinds of bacteria were found in this inflammatory tissue, but towards and beneath the peritoneum were colonies of micrococci occupying intervals between the cells and layer spaces probably lymphatic. This case is cited simply to point out what may be found in a case of advanced swine plague in which the lesions are concentrated in the large intestine chiefly, and the lungs mostly intact. It indicates somewhat the diffi culties in the way, the time that must be consumed before any trustworthy results are obtained, and the caution that must necessarily be exercised in coming to a conclusion.

In endeavoring to carry out the plan of isolating and cultivating the bacteria found in swine plague, the work was retarded by the difficulty of keeping on hand cases of swine plague contracted in the natural way, and by the heat of summer which completely interfered with the ordinary nutritive gelatine cultures. Finally a special "cold-box" was constructed in connection with the cold-air chamber of an ordinary refrig erator, and thickly padded with felt. The air within this box, in communication with that of the refrigerator by an upper and lower pipe, remained about 10°F. below the temperature of the laboratory, which for several months rarely fell below 90°F. at night. Plate-cultures and tubecultures were kept in this box, and only removed for purposes of examination and inoculat on. In this way only, were we able to utilize gelatine, after losing many plates upon which hours had been spent the day previous,

Perplexed by contradictory results, and failing to obtain any pathogenic germ by isolating the different forms found in the peritoneal effusion, the discovery of a fine bacillus in Germany, causing a disease in swine which was regarded as identical with swine plague in England and the United States, attracted our attention. The bacillus, of exceedingly small size, was described as being present in large numbers in the spleen and other organs of diseased pigs, and it was said that the disease could be at once determined by examining portions of the spleen, dried on cover glasses. As this bacillus had never been seen by us, we decided to examine the spleen very carefully, in order to determine its presence or absence.

The disease having for the moment died out at the experimental station, our attention was directed to an outbreak in Salem County, New Jersey, where it had assumed quite extensive proportions. Two young animals were killed. In both, the large intestine was extensively ulcerated, the lungs partially hepatized, and the large serous cavities filled with effusion. Without going into details we need but to state that in numerous cover-glass preparations of the spleen the fine bacillus was absent. Cover-glass preparations of lung tissue and of the serous effusions equally negative.

In regard to the cultures made in gelatine tubes, the results did not differ from those obtained heretofore. In three cultures of the blood of one animal, nothing grew; in one from the other animal, 3 or 4 colonies appeared. Several cultures from the same animal were entirely neg. ative. From the spleen of the first, 3 different microbes were isolated— 2 micrococci and a bacterium. In cultures from blood and serous exudates, several other forms were isolated. In all, 5 bacteria from these two animals were studied (4 micrococci and 1 bacterium). Inoculations of each into 2 mice and 2 pigs failed to produce any disease, with the exceptions mentioned below. The cultures in gelatine proved that both in the blood and in the spleen these bacteria were very few in numbers, so that they could not be detected in cover-glass preparations. We were fully satisfied that the fine bacillus claimed to be the cause of the disease in France and Germany was not present.

Anticipating somewhat the conclusions which we arrived at later concerning the real cause of this puzzling disease, we must say, at this point, that we no longer consider a micrococcus as the cause of all outbreaks of the disease known as swine plague. The first annual report of the Bureau mentions the death of 3 pigs from inoculation with a micrococcus. This micrococcus is easily distinguished by its peculiar growth on gelatine, which it rapidly liquefies. Since that time it was not found, excepting in one of the two cases just described. The inoculations with this produced a rise of temperature within ten days in the 2 animals, which subsided in a few days after. One of the animals was killed about seventeen days after inoculation. The lungs were found extensively hepatized. The presence of numerous lung worms left us in doubt as to the cause of this hepatization. Tubes inoculated from the spleen and blood remained sterile. The second animal died of swine plague one month after inoculation. The results which we obtained later on with another microbe lead us to suspect that this was a case produced by natural infection. Whether this micrococcus is a septic organism or one which is the cause of a definite disease in pigs cannot be answered at present.

The attention aroused abroad by Pasteur's vaccine as a protective against the disease known as rouget in France and rothlauf in Germany, and there regarded as identical with the disease prevailing in our own

country, led us to examine carefully two tubes of vaccine, known respectively as first and second vaccine, which were kindly sent to us by Prof. A. Liantard, of New York, and which he had received directly from Pasteur's laboratory. We were surprised to find that the microbe. in the vaccine was identical with that described by German investigators as being the cause of the disease in Germany. This microbe was without doubt a bacillus, exceedingly small to be sure, but not at all recalling the microbe originally described by Pasteur and Thuillier as having the form of a figure of eight. We shall later state our reasons for supposing that the microbe first discovered by Thuillier and the one. now cultivated as a vaccine in Pasteur's laboratory are two entirely dif ferent microbes and the cause of two distinct diseases. In the following chapter the results of our experiments with the vaccine are given in detail, and from them it will be seen that it does not prevent swine plague, for the simple reason that the vaccine of one disease cannot protect against another.

AN EXAMINATION OF PASTEUR'S VACCINE FOR ROUGET.

On October 16 a tube was received at the laboratory containing about 15 of Pasteur's vaccine for rouget, the European swine plague. The color of the liquid, which was faintly turbid, was of a light reddish yellow. A rubber cork securely closed the mouth of the tube. On drying minute portions on cover glasses for the purpose of examining the kind of microbe it contained, the culture liquid was found to contain a considerable proportion of solid matter, which formed a thick layer on the cover glass, and did not adhere firmly during the operation of staining and washing. This residue is in all probability peptone, as there is no precipitation on boiling. Judging from the amount of residue, the culture liquid contained at least 2 per cent.

When stained in methyl violet, prepared by adding a drop of an alcoholic solution to a watch glass of distilled water, and examined with a homogeneous objective, the microbes appeared as bacilli in the form of very slender filaments, which assumed various curves, loops, and broken lines. As the manner in which the vaccine had been put up did not seem to guarantee the absolute purity of the culture, a number of plate-cultures were made, whence to obtain pure cultures. Two days later a few scattered colonies appeared in the form of a rosette of club-shaped elements. These were small bacteria. On the following day a large number of very small round colonies could be discerned with a magnification of about 60 diameters. These were no doubt the fine bacilli. A greenish film began to encroach upon the plates and liquefy the gelatine. Five days after the plates had been prepared the colonies could not yet be distinguished with the naked eye. Under a 1-inch objective they appeared as large as pins' heads. To avoid total loss of the plates by the encroachment of the chromogenous, putrefactive microbe, a spot was selected under a dissecting microscope free from colonies of what were supposed to be contaminating bacteria, a minute portion of the gelatine layer containing a number of barely visible colonies was dug up and transferred to 10cc of beef broth with 1 per cent. peptone. On the 23d, two days after inoculation, a faint opalescence was observed in both tubes, which on shaking was resolved for the moment into delicate

A microbe was described by Schütz as being in the vaccine used in Baden which produced the same greenish coloration.

rolling clouds. There was no membrane or deposit. From another plate a tube culture in nutritive gelatine and one in beef broth with peptone were made in the same way. In three days the liquid culture presented the same appearance as those made from the other plate. Films, dried on cover glasses and stained for half a minute, contained the same bacilli as those found originally in Pasteur's liquid vaccine. The track of the platinum wire in the tube of gelatine became opaque in a few days. A row of cloud-like masses began to spread from it as a center and appeared as if strung on the needle track, which was now very faintly discernible (Plate II, Fig. 6). These clouds were fringed at the edges and when approaching one another, the entire growth presented the appearance of a minute test-tube brush forced down into the transparent gelatine. We were therefore led to conclude from microscopical and culture appearances that the microbe of Pasteur's vaccine was not a figure-of-eight form as he himself described, but a bacillus not to be mistaken for a micrococcus.

*

INOCULATION WITH THE FIRST VACCINE.

(a) In mice. It had been experimentally determined by Löffler that the bacillus of rouget resembles very closely the bacillus which produces septicæmia in mice in microscopic appearances, in its mode of growth in gelatine as well as in its behavior towards the white blood corpuscles in the body of the infected animal. If the bacillus of Pasteur's vaccine is identical with the bacillus of rouget in Germany, and not too attenuated, it should produce septicemia in mice. On October 17, three mice were inoculated by injecting, beneath the skin of the back of two, five drops; of the third, ten drops of the vaccine. They were kept in a large glass bell-jar, covered with a sheet of tin perforated with large holes over its entire surface, and were supplied abundantly with food and water. To avoid pain they were invariably chloroformed before inoculation. October 20 one mouse was plainly ill; it moved with difficulty, had a staring coat, and suffused, partly closed eyes. It was found dead the next morning, or about four days after inoculation. Portions of the spleen, liver, and blood from the heart were rubbed on cover glasses, dried and stained for half a minute in an aqueous solution of methyl violet. Two cultures, one in gelatine and one in beef-broth peptone, had been previously made. On the three cover glasses very fine bacilli were found quite abundantly, some free, and some within white blood corpuscles (Plate II, Fig. 5). In one cell, at least thirty could be counted. The presence of large numbers of bacilli in white blood corpuscles from the spleen of mice in which septicemia had been produced by the injection of putrid blood was pointed out as far back as 1878 by Koch, and lately comfirmed by Löffler and Schütz, in mice inoculated with the virus of rouget. The culture in gelatine of blood from the heart assumed precisely the same appearance as did the culture from the gelatine plates. The liquid culture proved to be made up entirely of the fine baccili, while its microscopic appearance coincided with the liquid cultures from the gelatine plates. October 24 another mouse showed symptoms of illness. The eyes were slightly suffused, the coat staring, and the respiration labored. It lived through the next day, but was found dead on the morning of the 26th, nine days after inoculation. In the liver and blood

*Within a few days a book on rouget (Lydtin u. Schottelius: Der Rothlauf d. Schweine) was received in which the term "test-tube brush" is also used in describing the appearance of the gelatine culture,

« ZurückWeiter »