Vermont. Results of the tuberculin tests of cattle in various States. Massachusetts Massachusetts, entire herds Connecticut.. New York, 1894 New York, 1897-98 New Jersey Illinois, 1897-98.. Illinois, 1899. Michigan Minnesota. Iowa Wisconsin: State. Experiment station tests Suspected herds.... Nonsuspected herds.. State veterinarian's tests Suspected herds Tests of local veterinarians under State veterinarian on cattle intended for shipment to States requiring tuberculin certificate... THE CHARACTERISTICS OF RABINOWITSCH'S BUTTER BACILLUS. The results of some of the earlier workers are open to criticism in view of Rabinowitsch's discovery of an acid-fast bacillus in butter morphologically similar to the tubercle bacillus. If guinea pigs are inoculated with milk or butter containing the acid-fast butter bacillus they may often die and will present lesions to the naked eye very similar to those produced by the tubercle bacillus. For that reason I give the following description of the cultural characteristics and post-mortem appearances caused by this organism taken from Annett's article. The characteristics of Rabinowitsch's micro-organism are as follows: It is immotile, and in form closely resembles the bacillus tuberculosis. The bacilli generally occur singly and are often slightly curved; but when growing rapidly in tissue bacilli are often found lying parallel. Sometimes they form long unbranched threads and sometimes are divided into short pieces. The bacilli are somewhat thicker than the tubercle bacillus and often show a club-shaped swelling on one side. Spores are not formed, but one portion of the bacillus stains often more intensely than the rest. The bacilli stained by many methods of staining tubercle bacilli can not be distinguished a Annett, H. E.: Tubercle bacilli in milk, butter, and margarine. Report Thompson Yates Laboratory, 1898-99, pp. 29-35. from the latter; only by the employment of very dilute watery solutions of methylene blue could any distinguishing feature be observed, viz, that bacilli from a culture of bacillus tuberculosis stain only at one spot, the rest of the bacillus remaining unstained; while in the case of bacillus pseudo-tuberculosis the whole bacillus stains faintly and generally uniformly, seldom showing a more deeply stained part. Cultural differences, however, occur. On agar, the bacilli taken direct from an infected animal produce visible colonies on the second or third day. At first the agar surface is covered with a thick, moist, creamy layer; in old cultures by a folded membrane often orange or copper colored. After repeated passages through animals cultures on agar or glycerin-agar show a dry, brittle, crumpled membrane resembling that of bacillus tuberculosis. In plate cultures the deep colonies are gray in color, round or oval, and uniformly granular. On the surface, colonies are better developed, have a uniform granular gray center, and a clear, wavy outer zone. The surface of the colony is often dry and conical. On butter-agar in fresh cultures the colonies are small, white, and dry, later spreading over the whole surface and becoming yellow or copper colored. On potato a luxuriantly growing, moist, gray layer is formed. In gelatin, growth proceeds very slowly at ordinary room temperatures, colonies becoming visible on the third day. In broth, and especially in glycerin broth, growth is rapid, forming in two or three days a folded membrane on the surface, the broth remaining clear, the culture closely resembling that of bacillus tuberculosis. Broth cultures are distinguishable from those of bacillus tuberculosis by their characteristic odor, being unpleasant and ammoniacal; that, of bacillus tuberculosis being agreeable and resembling the odor of flowers. A small quantity of indol is formed in broth cultures, which is not so in bacillus tuberculosis cultures. Milk is not coagulated, and on the surface is an abundant. yellowish-red layer which clings firmly to the glass. On albuminfree colorless media a growth appears in two or three days, becoming in ten days a thick, yellow, folded membrane; bacillus tuberculosis in the same time on such media forming a thin layer just covering the surface and just beginning to fold. The presence of fat in these bacilli can easily be demonstrated, as in the case of bacillus tuberculosis. PATHOGENIC PROPERTIES OF BACILLUS PSEUDO-TUBERCULOSIS. The following are the post-mortem appearances observed in a guinea pig killed three or four weeks after intraperitoneal injection of butter containing the bacillus pseudo-tuberculosis: There is a slightly distended abdomen; also peritonitis, with adhesions varying in nature from delicate fibrinous bands to firm connective tissue. The peritoneum and mesentery are studded with nodules. The mesenteric glands are swollen and may contain purulent or caseous matter. The liver is covered with nodules and patches which may be raised above the liver substance or may penetrate into the liver parenchyma. The spleen is sometimes only enlarged; at other times thickly studded with nodules. The kidneys show yellowish patches. The lungs are covered with small transparent nodules which do not penetrate into the lung tissue. The sternal lymphatic glands are swollen, but show no caseation. Numerous bacilli can be demonstrated in these lesions. Many animals after injection show considerable signs of illness during the first fourteen days, with diminution in weight, and then recover. Pure cultures of this bacillus are only pathogenic for guinea pigs (but not always); rabbits and white mice are immune. After intraperitoneal inoculation of guinea pigs some die in from four to eight weeks, with considerable emaciation, and show the following post-mortem appearances; others survive: At the seat of inoculation there is a purulent infiltration containing the characteristic bacilli; also peritonitis, varying in intensity from a flocculent fibrinous exudation to strong connective tissue adhesions. The mesentery is studded with small nodules; the glands are enlarged, but not caseous; there are patches on the liver, and miliary nodules throughout an enlarged spleen. The thoracic cavity and organs are often almost exempt from lesions. Infected animals do not react to tuberculin. Histologically the nodules in the liver and spleen consist of a collection of lymphoid elements with but very few epithelioid and multinuclear cells. The bacilli are found in the middle of a young nodule, toward the periphery if caseation has commenced. The typical giant cells of tuberculosis do not occur. More often-especially after inoculation with butter containing the pseudo-tuberculosis bacilli-the nodules appear to consist of a central necrosed portion surrounded by a leucocytic infiltrated area. COLLECTION OF SAMPLES AND TECHNIC. The samples of milk were all collected and brought to the Hygienic Laboratory by an inspector of the health department of the District of Columbia. Usually a pint bottle, though sometimes a quart, with the paper cap untampered with was obtained either from the diary or delivery wagon. The bottle was at once placed on ice by the collector and usually reached the laboratory in about one hour after collection. A few samples were obtained from some of the hospitals and charitable institutions of the District. The milk and cream were well mixed by vigorously shaking the bottle. The sample for plating was taken out with a sterile pipette, and then 50 cubic centimeters of the mixed milk was put into a large sterile centrifuge flask. To 24907-Bull. 41-08-12 the 50 cubic centimeters of milk was added 100 cubic centimeters of sterile water. The flask was then put into the centrifuge machine and centrifuged for one hour at about 2,000 revolutions per minute. The milk was diluted with twice its volume of water with the idea that it would decrease the specific gravity of the milk and so permit of the easier sedimentation of the tubercle bacilli. Usually only one animal was inoculated from each sample, though in some cases two animals were used. Guinea pigs, largely those raised in the laboratory, of as uniform weight as obtainable, were inoculated with 5 cubic centimeters of the sediment of this centrifugalized mixture of milk and water. The inoculation was made subcutaneously in the belly wall. For each guinea pig a different syringe was used. All of the guinea pigs, usually 8, that being the usual number of daily samples, inoculated on the same day were kept in the same cage, those that remained healthy being controls on their environment, etc. The guinea pigs were examined for enlarged glands after about four weeks, and those with enlarged glands were separated from the others so as to avoid the danger of infecting others if the glands broke down. Many of the animals inoculated died from acute infection with the millions of other bacteria in the milk. Autopsies were made on all the animals that died, but no attempt was made to determine the causal organisms other than the tubercle bacillus. Those guinea pigs which did not die in at least two months were chloroformed, after having been tested with tuberculin, and careful autopsies were made on each animal. Smears, cultures, and sections were made from the various organs of the animals that showed any change from the normal. The smears were stained with carbolfuchsin and examined for acid-fast bacilli. Cultures were made on glycerinized potato and glycerin-agar. In no instance did any of the cultures show a quick-growing acid-fast organism resembling in any way Rabinowitch's butter bacillus. The sections were stained with carbol-fuchsin for tubercle bacilli, and also with hæmalum and eosine for histological appearances. The above details were carried out with few exceptions in all of the animals that gave a positive result. It occurred to me that those animals which had tuberculosis might be differentiated from those with other infections by giving all of the guinea pigs alive at the end of two months a sufficient dose of tuberculin to cause the death of the tuberculous animals in less than twenty-four hours. Several preliminary tests on known tubercular animals showed that 2 cubic centimeters of crude tuberculin given subcutaneously would almost invariably cause the death of such a guinea pig in from six to eighteen hours. As high as 7 cubic centimeters of the same tuberculin given to a healthy pig caused only a temporary discomfort, passing off in a few hours. A rather hasty search of the literature failed to show that this idea of giving an amount of tuberculin sufficient to cause the death of a tubercular animal as a means of differentiating true tuberculosis from infection with other acid-fast organisms had ever been used by previous workers. The febrile reaction in a sick guinea pig on account of the great variation in the temperature of the animal from handling, etc., is too variable a factor, and a more definite reaction, such as the death of the animal, is necessary. The technic was as follows: All of the animals, in lots of about 30, were given early in the morning 2 cubic centimeters of the tuberculin subcutaneously; they were closely watched and as soon as an animal appeared sick it was placed aside; as soon after death as possible the animal was autopsied; smears, cultures, and sections were made. Of all the guinea pigs, about 250. that received the tuberculin, no animal that did not have tuberculosis died. Two or three that had slight lesions did not die, but became sick. It was noted that all of the animals died whose lesions had caseated. The reaction, I think, was of distinct service in eliminating infections with other acid-fast organisms. The suggestion is made that with some modification the procedure may have a distinct. place as an aid in differentiating true tuberculosis from infections with other acid-fast organisms which produce tubercular-like lesions. Samples of milk were examined from 104 different dairies; 10 samples from 7 hospitals and asylums are also included in this number, they being charged also to the dairy supplying the milk. The following tables show the laboratory number of the dairy, where collected, date of collection, whether the guinea pig inoculated died or was killed, interval between inoculation and death, and results of the autopsy. It is interesting to note that where 2 guinea pigs were inoculated with the same sample of milk, in two instances both animals showed tuberculosis and in two instances only one was positive: |