diseased cattle, for which $7,069 was paid, being an average of about $23 per head. In Maryland the quarantine has been made very efficient by placing a chain, fastened with a numbered lock, around the horns, or, with hornless cattle, around the neck of every exposed animal. This has prevented the substitution of one animal for another, and it has also led to the prompt detection of any quarantined cattle which have been allowed to stray beyond the boundaries of the infected premises. The sick animals have been promptly slaughtered, and it is believed that the good effects of this work are already seen in the decreased number of new herds infected. In a number of cases where infected herds have been of unusual danger to surrounding cattle this Department has purchased and destroyed the sick animals, and the State has then condemned and slaughtered the remainder of the herd, thus entirely eradicating the disease at once. Unfortunately the State appropriation has not been large enough to do this in as many cases as seemed desirable. The No recent investigations have been made in Pennsylvania. governor's agent, Mr. T. J. Edge, reports that during the year ending November 30, 1886, 128 diseased animals were condemned and slaughtered. LESIONS FOUND IN AMERICAN PLEURO-PNEUMONIA. Many persons who have not had an opportunity for the post-mortem examination of animals affected with pleuro-pneumonia, have expressed doubts regarding the nature of the disease in this country, some considering it to be tuberculosis, while others have gone so far as to speak of it as blood poisoning with abscesses of the lungs. To make it clear that the disease in America is identical with the pleuropneumonia of Europe, and that the lung lesions are neither the result of tubercles nor abscesses, plates X, XI, and XII have been prepared from actual specimens of lungs encountered in the outbreaks previously referred to. Plate X is a drawing of a lung from a steer belonging to Mr. Nels Morris that died in the Phoenix distillery stables at Chicago. The lung was photographed and the photograph colored in Chicago, and the plate was made by Dr. Marx, the Department artist, from this colored photograph. Plates XI and XII were made direct from fresh specimens removed from the carcasses of cows slaughtered in Baltimore. Plate XI shows an early stage of a very acute attack, in which the lesions consist of distension of the interlobular connective tissue with lymph, infarction of a portion of the lobules, and pleuritis, with a great abundance of false membranes. Plates X and XII show more advanced cases in which the lung is completely hepatized, different lobules showing different stages of inflammation, the spaces of the inter-lobular connective tissue being filled with lymph and the pleura greatly thickened. These lungs presented no appearance whatever of tubercles or abscesses, which is true of nearly all lungs we have examined in acute attacks of this disease. INVESTIGATIONS OF SWINE DISEASES. In view of the results of investigations which have shown the existence of two distinct infectious diseases in swine, perhaps of equal virulence and distribution, a change in the nomenclature becomes necessary in order to avoid any confusion in the future. Since these two diseases have been considered as one in the past, and the names swine-plague and hog-cholera have been applied indiscriminately, we prefer to retain both names, with a more restricted meaning, using the name hog-cholera for the disease described in the last report as swine-plague, which is produced by a motile bacterium, and applying the name swine-plague to the other disease, the chief seat of which is in the lungs. This change is the more desirable since recent investigations have shown that the latter disease exists in Germany, where it is called swine-plague (Schweineseuche). INVESTIGATIONS OF HOG-CHOLERA. Some additional biological facts concerning the bacterium_which produces the disease.-In the second annual report of the Bureau and the Annual Report of the Department for 1885, the bacterium of hog cholera was quite minutely described, so that no one acquainted with bacteriological investigations would find it difficult to recognize it when found. The descriptions of size, shape, and mode of staining referred to cover-glass preparations made from the blood and the internal organs directly. These characters change somewhat when the bacterium is cultivated in artificial media. Thus the bacteria grown upon potato vary slightly in size and appearance from those obtained from meat infusions and from nutrient gelatine. On the other hand, their appearance is the same whether the spleen of mice, rabbits, guinea-pigs, or swine be subjected to microscopic examination. The microbe was characterized as a motile bacterium 1.2 to 1.5 micromillimeters long and .6 micromillimeter broad, growing readily in neutralized and even slightly acid meat infusions, milk, on potato, and gelatine which is not liquefied. During the past year the bacterium has been studied very carefully, with a view to determine the best means of preventing its multiplication, and thus preventing the spread of the disease itself. The conclusions arrived at are given in full below, but will be summarized from a practical point of view in the chapter on prevention. Growth of the bacterium in simple hay infusion.-This was prepared by allowing finely cut hay to soak in water for three or four days, filtering off the amber liquid and sterilizing. Two tubes were inoculated with a drop from cultures in meat-infusion peptone at different times. In both the following features were observed: There was a slight turbidity within two days, which did not deepen perceptibly. The bacteria were somewhat larger than in more nutritive liquids. In the shorter forms there could be seen at each extremity more refrangent spherical masses, while the central portions of the rod seemed empty. Longer rods contained three or four of these bodies. When stained they appeared darker than the rest of the rod. They were consequently not spores, but very probably masses of protoplasm, which had contracted into these globules, and which indicated a degeneration of the bacteria. There were also forms present which were beaded, club-shaped, and distorted. Though the acid hay infusion is not a suitable medium, yet the bacterium of hog-cholera evidently multiplies in it to some extent, and we may infer that in any organic infusions, such as are formed about pens among the food of swine, the bacterium may multiply under the influence of a hot sun and be afterward taken into the system with the food and water. Multiplication of the bacterium in water.-The hardiness of this microbe is well illustrated by its capacity for multiplication in ordinary drinking water. To determine this the following experiment was made: September 8: A culture tube containing very clear Potomac drinking water,* which had been sterilized several weeks previous by a temperature above 110 ̊C., was inoculated from a pure culture of the bacterium. By mixing a given quantity of this water immediately after inoculation with gelatine, and making a plate culture of the same, it was found that the water contained about 26,240 bacteria in 1o. The water was kept in the laboratory, in which the temperature corresponded closely with that prevailing out-doors. It was examined from time to time on gelatine plates, and the number calculated for 1cc. The following figures give the results obtained: September 8: 26,240 in 1cc (immediately after inoculation). September 9: 201,600 in 1cc September 10: 1,296,000 in 1cc. September 11: Too numerous on plate to be counted. September 13: 2,608,200 in 1cc. September 15: 1,519,560 in 1cc. September 17: 1,306,308 in 1cc. September 29: 83,700 in 1cc. October 12: 19,125 in 1cc. November 18: 225 in 1cc. December 6: A few bacteria still present, as determined by liquid cultures. January 11: No growth on plates. That the bacterium can be kept alive in clear river water for four months and perhaps longer is a fact very significant in itself. When we consider, moreover, that the added bacteria multiplied so that each individual was represented by ten at the end of five days, the hardiness of the bacterium is very evident. The danger from infected streams into which feces from sick animals find their way is thus proved beyond a doubt. The effect of simple drying on the bacterium of hog-cholera.-The resistance of this microbe to various agencies, physical and chemical, is likewise of considerable importance in determining the manner of infection, the spread of epidemics, and the possible means within reach of destroying the virus. In order to test its vitality when deprived of moisture the following experiments were carried out: January 19, 1886: A number of cover-glasses were heated in the Bunsen flame and then placed on a flamed glass plate under a flamed funnel. The mouth of the funnel was plugged with cotton wool to allow desiccation while excluding aerial organisms. When cool a drop from a pure liquid culture of the bacterium was placed on each cover with a pipette, and the whole left in the laboratory at a temperature of 65° to 80° F. The culture used had been prepared January 7 from the fifth spleen culture, hence was twelve days old. January 21: Two tubes of nutritive liquid inoculated by dropping a cover-glass into each. Both turbid next day, containing the bacterium of hog-cholera only. January 25: Two tubes inoculated in the same way. Same result next day. January 28: Two additional tubes receive each a cover-glass. They were still clear on the following day. January 29: Two tubes inoculated. January 30: One tube. These five tubes remained permanently clear. In one, inoculated January 29, a fungus had developed from the cover-glass in the bottom of the liquid. This, however, remained clear. *When drawn this water did not contain more than 100 to 200 bacteria to the cubic centimeter. This series placed the death-point of the bacterium between the seventh and the ninth day. A second series of covers received each one drop from a culture obtained from a mouse which had died from the effect of inoculation. The culture in beef infusion peptone was ten days old. Treated in the same manner as in the preceding experiment, the bacteria were found to resist drying for ten days, when the stock of cover. glasses was exhausted. To determine whether bacteria in the body of the diseased animal possess a greater power of resistance than those in cultures the following experiments were made: Some bits of the spleen of a pig which was found crowded with the specific bacteria of hog-cholera were dried on sterile cover-glasses as above described, and then dropped into tubes containing beef infusion. Cover-glasses which had been dried for from eight to sixteen days were able to develop pure cultures of the bacterium in the tubes. The stock of covers being exhausted, another series was tried in the same way. The blood of spleen tissue was permitted to dry undisturbed until the seventh day, when the first tube was inoculated. Cover-glasses dropped into cultures on the seventeenth, nineteenth, twentieth, twenty-first, twenty-fourth, and twenty-sixth days left the cultures sterile. Those dropped in on the eighteenth and twenty-second days produced pure cultures of the bacterium. These experiments indicate a greater resistance of the bacterium in spleen tissue, which may live twentytwo days in a dry atmosphere at a temperature of 70° to 80° F. On May 8 five cover-glasses upon which bits of spleen tissue, known to contain the bacterium of hog-cholera, had been dried under a plugged funnel since March 20, were dropped into tubes of beef infusion. On the following day all tubes were turbid. In one of them bacillus subtilis was present. All the others were pure cultures, as determined by microscopical examination. Two of these were tested furthermore on gelatine plates with the same results. This indicates that in the varying temperature of a room desiccation of small bits of tissue (not so large as a pin's head) failed to destroy the bacterium in forty-nine days. In the experiments with dried cultures those ten and eleven days old were chosen, so that if any resistant spore state did form in liquids it would be present. It is highly probable, however, that if cultures but a few days old had been chosen the bacteria would have resisted drying much longer. These experiments give the following results: A liquid culture eleven days old resisted drying for nine days; another, ten days old, at least ten days. Bacteria in tissue may resist destruction after drying for from twenty-two to forty-nine days. The method of drying the bacteria on cover-glasses and introducing the latter into liquid does not inform us whether most bacteria die within the same time or whether some resist much longer than others. Hence the following expedient was resorted to, which Koch had introduced in the study of disinfectants: Silk threads sterilized by boiling several times in distilled water were dried and steeped in a beef infusion peptone culture about one week old. The culture containing the threads was allowed to dry in the incubator for one day, then placed in a sterilized bottle plugged with cotton. Each day, beginning with the second, one or two threads were placed in a layer of nutritive gelatine on a glass plate so that the thread was completely covered by the gelatine. Characteristic colonies of the bacterium appeared around the thread within two days, though the plates were usually kept under observation five days. For twenty-one days isolated colonies and groups of colonies appeared in moderate abundance on the threads, when the stock of the latter was exhausted. In another similar series the threads were laid upon a sterile plate and a twentyfour hours' liquid culture poured upon them and allowed to dry uncovered in the incubator for one day. These threads, still undisturbed on the plate, were placed in the laboratory, covered with a bell glass. On the fifteenth day the testing began, a single thread being placed in the gelatine layer each day for sixteen days. Colonies of the bacterium developed in large numbers until the twenty-second day, when they diminished in number. On the twenty-seventh and twenty-eighth days no colonies appeared. On the three following days a few appeared, when the series was closed. The bacterium of hog-cholera may therefore remain alive, during continuous desiccation, for from ten days to nearly two months. The variation in the results obtained is no doubt due to the different vitality of the cultures used. The gelatine-plate method is not so delicate a test as the method of liquid cultures, as it would be difficult to tell when the last bacterium died, a single colony under the thread escaping observation very easily. A single bacterium would invariably reveal its presence in a liquid after a time by multiplication. For the same reason the latter method needs greater care; the liquid cultures must be examined microscopically, and if there be any doubt 'still remaining they must be tested on gelatine; for a single foreign microbe gaining access to the culture tube might introduce an error into the results, which is easily avoided on the gelatine plate by observing the characters of the colonies. It had been determined by a large number of experiments that cultures of the bacterium of hog-cholera can be sterilized-in other words, that the bacterium itself may be destroyed-by an exposure to 58° C. for from 15 to 20 minutes. To determine whether dried blood or spleen tissue containing the bacterium was more resistant the following experiment was tried: Spleen pulp from a case of hog-cholera was rubbed upon sterile cover-glasses and allowed to dry under a plugged funnel for 24 hours at a temperature of 65° to 75° F. Four tubes of beef infusion, after a cover-glass had been dropped into each, were exposed to a temperature of 58° C. for 15, 20, 29, and 41 minutes, respectively. These remained permanently sterile, while a fifth tube, which had been inoculated in the same way but not heated, contained on the following day a pure culture of the hog-cholera bacterium. It should be added that each cover-glass contained a considerable number of germs, according to microscopic examination of different parts of the spleen. The bacterium within the body of the diseased animal cannot therefore be regarded more resistant than when cultivated in liquids. Effect of boiling water.-Culture tubes containing about 10cc of meat infusion were placed in boiling water until at the boiling-point. They were then removed, and a sterile cover-glass, upon which a bit of spleen tissue had been drying for five days under a plugged funnel, was dropped into each tube. These were immediately placed in ice water. A preliminary experiment had shown that the temperature in these tubes fell below 40 C. in less than a minute. The spleen had been previously found to contain the bacteria in large numbers. Of four tubes treated in this way two became turbid with the specific bacterium; the others remained sterile. In a subsequent experiment, four tubes were inoculated near the boiling temperature and one as a check. This latter developed into a pure culture of the bacterium; the heated tubes remained permanently clear. An almost momentary exposure of the dried bacteria to boiling water is sufficient, therefore, to destroy their vitality. Resistance to various chemical substances or disinfectants.-In the following experiments on the effect of various agents on the vitality of the bacterium of hog-cholera the methods used by Koch were not adopted, because liquids are far more sensitive to bacteria than solid media. A single colony upon gelatine, the descendants of a single germ, may escape the eye, but the same microbe in a nutrient liquid would cloud it within a few days. There is, to be sure, for this very reason, greater danger in the use of liquid media, since the introduction of a single foreign microbe might lead to the same conclusions as the introduction of a dozen or a hundred, while a few bacteria accidently caught on the gelatine would lead to no errors of interpretation. The results obtained by the method given below were so uniform, the absence of contamination was so constant, that we can recommend it in all similar determinations. The disinfectant solution was diluted with sterile distilled water in a test-tube or watch-glass previously sterilized by heat. A few drops from a pure culture of the hog-cholera bacterium were mixed with 4 or 5 of this dilution, and a minute portion transferred at given intervals, by means of a platinum loop, to culture tubes containing beef broth. These tubes were then placed in a temperature of 95° to 100° F., where they remained from one to four days. Tubes which remained clear at the end of this period were sterile, as shown by numerous tubes which were watched for several weeks. |