Bulletin No. 13. U. S. DEPARTMENT OE AGRICULTURE. SC HTTKKATT OF ANLMAL 1XI)U8TKY. /' , -rs %0% TUBERCULOSIS INVESTIGATIONS. THE GROW'I'K OF TH1-: TirRERCULOSLS BACILLUS UPON ACID MEDIA. liy E. A. ]>;•: Schwkinitz and Mahion Douskt. |FUKTHEK EXPERIMENTS WlPIf AN ATTENUATED TUBERCrUOSIS BA- CILLUS. By E. A. i)K ScuwKixiT/ and E. C. Schhoedkk. |THE EFFECT OF TUBERCULIN INJECTIONS UPON" THE MILK OF HEALTHY AND DISEASED COWS. ]>y E. A. i>K ScuwKixiTZ. Prepared under the direction of Dr. D. E. SALMON, Oiiel" of "tli«i Bxjii'eavi <>r ^^iiimal InK SCHWKINITZ. Prepared iiiuk'i- the (lirection oC Dr. D. E. SALMON, Issued September 19, 1896. WASHINGTON : GOVERNMENT PRINTING OFFICE. 18 6. 1^ ^^ net 20 »«* -4 LETTER OF TRANSMITTAL. U. S. ])epartment of Agriculture, Bureau of Animal Industry, WaHhhujton^ D. C, July 15, ISOd. Sir: I hiiv^e the honor to trausmit herewith, and to recommend for publication as a bulletin of this Bureau, the manuscript containing a report of investigations upon the cultivation of the tuberculosis bacil- lus ui^on acid media, and of some further experiments with reference to the effect of tuberculin upon the milk of cows. These experiments have been conducted under the immediate super- vision of Dr. E. A. de Schweinitz, assisted by Drs. Marion Dorset and E. 0. Schroeder, and the publication of the results obtained will furnish a (convenient and desirable method of preserving and distributing the results of scientific investigations in this important part of the work of this Bureau. Very respectfully, D. E. Salmon, Chief of Bureau. Hon. J. Sterling Morton, /Secretary of AyricuHure. 3 LETTER OF SUBMITTAL. U. H. Department of Agriculture, Washington, I). C, July ], 1896. Sir : 1 liercwitli submit for i)ublication some results of investigations upon the cultivation of the tuberculosis bacillus u})on acid media, tlie innocuous character of an attenuated tuberculosis bacillus, and some further experiments Avith reference to the effect of tuberculin upon the milk of cows. Charts showing the average temperature reactions of cattle as regards breed are also appended. Kespectfully, E. A. DE SCHWEINITZ, Chief Biochemic Division. Dr. D. E. Salmon, Chief of Bureau of Animal Indns'ri/. i COIsTTEI^TS. Paga Tlie crowtli of the tuberculosis bacillus upou acid inedia. By E. A. do Scliweinitz, Pli. D., M. D., aud Mariou Dorset, M. D 7 Introduction 7 Observations 8 Detn ils of experiments 9 Further experiments with an attenuated tuberculosis bacillus. ]?y. E. A. de Schweinitz, Ph. D., M. D., and E. C. Schroeder, M. D. V 11 Experiment with a monkey 11 Results of guinea pig inoculations 12 The effect of tuberculin injections njion the milk of healthy and diseased cows. By E. A. de Schweiuitz, Ph. D., M. D 15 Analyses of nulk of cows injected with tuberculin 18 Temperature reactions of the cows used in the milk experiments lil ILLUSTRATIONS. TLATES. Pago. PI. 1. Bacillus tuberculosis upon normal and acid media ^( 2. Bacillus tuberculosis upon acid mettia coutaining free bydrocbloric acid, aud upon artificial media 9 FIGURES. Fig. 1. Average temperature of 8,930 healthy and 1,191 diseased cattle injected with tuberculin 21 2. Average temperature reaction of Jersey cattle 22 3. Average temperature reaction of Holstein cattle 23 4 . Average temperature reaction of Durham cattle 24 5. Average temperature reaction of Ayrshire cattle 2") G. Average temperature reaction of Guernsey cattle 26 7. Average temperature reaction of Grade cattle 27 6 TUBERCULOSIS INVESTIGATIONS. THE GROWTH OF THE TUBERCULOSIS BACILLUS UPON ACID MEDIA, By E. A. i>E SciiWKiNiTz, Ph. D., M. D., and Makion Dohset, M. D. INTRODUCTION. In the Pliiladelpliia Medical Kews of December 8, 1894, one of us (De Scbweiiiitz) published au article upon the attenuated bacillus tuber- culosis and its effect upon guinea pigs, and called attention to the fact that the tuberculosis germ would grow satisfactorily upon media having au acid rea(!tion. This ai)peared to be an important observation, and while we have for several years been cultivating the tuberculosis germ upon various media, some interesting facts have been noted which are Avorthy of record. Pawlosky, Ann. de I'lnstitut Pasteur, 1888 (and Sander) (xVrchiv fiir Ilyg., I>d. XVI, p. 238) have shown that vegetable broths may be used for the cultivation of the bacillus tuberculosis, and (in the Centralb. fiir Bak. und Parasit., August 8, 1895, Nos. 4, 5) Lubinski describes the utilization of this acid broth in the preparation of solid and liquid media, either with or without the addition of beef broth. Our cultures have been made upon the ordinary i)eptonized meat broth prepared from fresh meat and upon the artificial media, which, as one of us noted earlier, has been very useful in studying the products of germs. We tind that the tuberculosis bacillus will grow upon acid N beef broth which requires 21.G c. c. ^0 sodium hydrate solution to neu- tralize every 100 c. c. of the media. It will also grow upon acid arti- jST ficial media every 100 c. c. of which lequires 45 c. c. of ^,. sodium hydrate solution for neutralization. This acidity in some instances was due to the acids iu the meat. In the artificial media it was due to the acid phosphate of potassium used. We have also added free hydrochloric acid to the acid beef broth N and to the acid artificial media, so that from 1 c. c. to 3 c. c. ^^ hydro- chloric acid have been contained in every 100 c. c. of the media. The presence of free hydrochloric acid was tested, both before and after the sterilization of the media, and after the bacilli had begun to multiply, by means of cougo red or phloroglucin and vanillin. The congo-red 7 8 test was not satisfactory in tlie beef brotli, tlie albuminoids and pos- sibly organic acids i)resent evidently interfering- with tlie reaction. The phloroglucin and vanillin test, however, proved conclusively the presence of free hydrochloric acid in the media, upon which there was a good growth of the tuberculosis bacillus. The exact quantity of the free hydrochloric acid present in these cultures could not be readily determined, as when hydrochloric acid was added to the media a por- tion apparently first decomposed the organic salts and phosphates. After the growth of the tuberculosis germ is well advanced upon the media described and has apparently ceased, the acid reaction of the cultures is still more marked, requiring for the neutralization of the acid substance produced by the germ to every 100 c. c. of cul- N ture 12.0 c. c. to 15 c. c. ^,. sodium hydrate, Lubiuski notes a decided variation in the morphology of the germ as grown upon the acid vegetable broth. The bacilli, he says, often appear in long fdaments, having, however, the same thickness that the germs usually possess. The filament forms described by Metschnikoff, Fischel, and Jones varied in thickness, showed side chains, and were not joined, while Lubinski reports characteristic streptococci-like for- mation. Jones could find this filamentous growth only after the cul- tures were four to five months old, Lubinski observed it in ten days. OBSERVATIONS. Our observations upon the character of the germs grown upon acid beef broth media and their mori^hological changes are somewhat differ- ent. Instead of a thread-like growth or chain growth Ave have often found what appeared to be spores, and sometimes the bacilli seem very much thicker and longer than the ordinary germs. In one culture examined the germ had been from generation to generation during four years grown on acid media and might be expected to show Lubinski's chain-like formation, but instead there was an apparent spore formation perceptible. The appearance of the germs grown u])on our acid media can be seen from the accompanying plates. PI. I, fig. 1, shows the bacillus grown upon glycerin beef broth for four mouths; Neisser's spore stain. PI. I, fig. 2, shows the bacillus grown for forty generations upon an acid media. The filament forms are not observed. There is a slight thickening of the bacillus and rounding at the end often noted, which would appear to be spore formation. There is a different change in the morphology of the germ as grown upon our media which confirms the conclusions of Lubinski that we have heteromorphic forms of the tuberculosis bacillus and a genuine pleomorphismus caused by the changed conditions of life. Bruns (Centralblatt flir Bak. und Parasitenkuude, Bd. XVII, Abth. i, Ko. 23) describes a germ which gives Keisser's spore reaction and in that particular eviilently corresponds to the ones we have pictured. Bui, 13, Bureau of Animal Industry. Plate I. * . / t <• 1 ■\ Fin. 1.— Seventh generatiou in glycerine beef Inoth. Neisser's -spore .stain. ( x ■~'0U0.) ' i 1 * s t *> • * • / - Fig. '-i. Foitietli yreneiatiou. ai-iil media. Neisser's .si)i ire stain. ( ^ :200i». i Haines, del. Bacillus tuberculosis upon Neutral and Acid Media. Bji 13, Bureau of Animal Ind'jstry. PLATE II. Haines, del. \ i ^ Fig. 1.— Culture foutaining free hydrochiijric acid. Neisser's spore stain. ( x 2000. ) if%. -^z" » V \ Fig. :;. — Artificial culture. Neisser's spore stain. ( X aOOO.) Bacillus tuberculosis upon Acid Media containing Free Hydrochloric Acid, and UPON Artificial Media. 9 PI. TI, t\g. 1, sbows the germ grown ui)oii acid media containing free bydrocbloric acid. PJ. II, fig. 2, is a drawing of the tuberculosis germ grown on arti- ficial media. We do not here observe quite the same change in the morphology of the germ. DETAILS OF EXPERIMENTS. A more detailed description of our experiments may be interest- ing. The reaction of the media prepared for the cultivation of the tuberculosis germ was not changed in the iirocess of sterilization. A quantitative check of the amount of acid was made before and after this ])rocess. First experiment. — The media used for this experiment was alkaline and required ibr neutralization 1 c. c, <,, hydrochloric acid for every 50 c. c. of the media. a. To each of 2 flasks 50 c. c. each, 5 c. c. ^7., NaOII were added. b. To each of 2 flasks aO c. c. each, 20 c. c. j^j, NaOH were added. N c. To each of 2 flasks 50 c. c. each, 1 c. c. ^, HCl were added. The six were inoculated at the same time with tuberculosis bacilli from (A 14). Within two weeks a good growth of the germ was noted over the surface of flasks a and c, while b showesks 101) c. c, 2 c. c. ^^ HCl were added. c. To each of 2 flasks 100 c. c, .'> c. c. ,^, HCl were added. These flasks were all inoculated with a virulent germ, and although it required several weeks for the growth to begin, after four weeks there was growth in all of the cultures, the most acid ones seeming' to contain as good a growth as the others. Every 100 c. c. of the a media IST had required 11. G c. c. ,^, NaOH. Every 100 c. c. of the b media had required IG.C c. c. -. >, NaOH. N Every 100 c. c. of the c media had required 21.G c. c. jt^, NaOII for neutralization. Fourth experiment. — The media used in this experiment was the artificial media described by one of us — De Schweinitz — in 1S02, and it N required 45 c. c. ^^^ NaOH lor every 100 c. c. for neutralization. a 100 c. c. one flask of the media as noted. h 100 c. c. one flask of the media as noted with the addition of 2 c. c. ^, hydrochloric acid. These were inoculated from another artificial culture, and both within fifteen days showed a good growth, the one containing hydro- chloric acid showing a slightly better growth than the other. Drs.Trudeau and Baldwin have pointed out that the apparent retarda- tion of the growth of the tuberculosis bacillus by the acid reaction of the media can be overcome by neutralization. This is true; but we have also found that if the germ is transferred from an acid culture after it has been cultivated on acid media for a number of months to a neutral nutrient fluid it will no longer grow. There would seem from this to be probably a poisonous principle secreted by the gei m. It may be a true acid or, more likely, a substance with an acid reaction. In the experiment, as the germs used for inoculation still floated on the surface of the culture they should have been alive unless some sub- stance especially inimical to their growth was i^roduced in the cultures. These experiments show clearly that the tuberculosis bacillus can be readily accustomed to an acid nutrient fluid, that it can easily adapt itself to the changed conditions of life, and that even a small amount of free hydrochloric acid does not interfere with its growth. They tend to show further, we think, that under certain conditions there is probably a poisonous substance produced by the germs which is inimical to their own life. The adaptability of the bacillus to a variety of media can best be explained by spore formation. March 11, 189G. FURTHER EXPERIMENTS WITH AN ATTENUATED TUBERCULOSIS BACILLUS. By E. A. DE ScHWKixiTZ, Ph. D., M. D., and E. C. Schroedeu, M. D. V. Ill the Medical News, December 8, 1894, one of us (E. A. de Scbwei- iiitz) briefly described an attenuated tuberculosis bacillus (human), and noted that while originally very virulent, by a special method of culti- vation it had been rendered very harmless, and not only failed to pro- duce disease in guinea pigs and rabbits, but in some instances imparted to animals inoculated with it a distinct resistance to tuberculosis upon a subsequent inoculation with a virulent germ. The experiments with this attenuated germ have been continued, and we desire to present further evidence of the apparent absolutely harmless character of this attenuated germ, reserving for a later article further reports of its immunizing properties. EXPERIMENT WITH A MONKEY. After a number of guinea pigs and cattle had been inoculated with this attenuated germ it seemed desirable to test its effect upon an ani- mal susceptible to tuberculosis and more like man. Accordingly a monkey placed at our disposal by Dr. Kinyoun was inoculated on September 10, 1895, with l c. c. of an emulsion of a tubercle culture thirtieth generation. The inoculation was made from a culture in which there was an active growth. On September 28, 1895, the injection with this material ^ c. c. of tuberculosis culture thirty-second generation was repeated, and again on January 20, 189G, 2 c. c. of tubercle culture thirty- tliird generation. At the seat of the first and second injections small nodules were formed, while no local lesion was noted at the seat of the third injection. On April 9 the monkey was found dead. It had not been well for three weeks and was quite sick during the five or six days immediately preceding death. It had no appetite, the hair was rough, there was nausea and great thirst and evidence of distress. Post-mortem exami- nation revealed congested lungs and an inflammatory condition of ileum and caecum. Dr. Schroeder says "there were no signs of tuber- culosis and the small nodules formed at the time of the first two injections had become absorbed." This attenuated germ was, there- fore, innocuous to the monkey, though, as noted before, it had been obtained from an originally very virulent germ. 11 12 REST'LTS OF GTTINEA PICI INOCULATIONS. The guinea i)ig iiioculatioius made by one of us (Scliroeder) witli this attenuated germ to test its virulence were as follows: November 24, 1S04, Nos. 280, 281, 282, and 283 received each an abdominal injection subcutaneously of ^ c. c. tubercle culture twenty-third generation. On January 25, 1895, each pig again received 1 c. c. tubercle culture twenty-sixth generation. October 7, 1895, guinea pig No. 281 was found dead. A small dry, cheesy nodule at the seat of one injection was noted, but there was no other evidence of tuberculosis, and death resulted from inflammation of the bowels. On January 3, 189G, guinea pig No. 282 was found dead from pneu- monia. There was no evidence of tuberculosis. The other two pigs, Nos. 280 and 283, are alive and well at this time. Guinea pigs Nos. 55, 5G, 57, 58, and 59 were inoculated on October 2, 1895, subcutaneously with 1 c. c. tubercle culture thirty-second gen- eration. On October 17 guinea pig No. 55 was found dead, and post- mortem revealed a general inflammatory condition of subcutaneous tissue over entire abdomen. The other animals, Nos. 50, 57, 58, and 59, are alive and well at this writing. Again, January 25, 1895, guinea pigs Nos. 331 and 332 were inocu- lated with i c. c. tubercle culture twenty-sixth generation. On Decem- ber 30, 1895, guinea pig No. 331 was found dead from pneumonia. The liver had a peculiar mottled appearance and a few yellowish patches. Though a coverglass preparation showed no tubercle bacilli, two pigs, Nos. 121 and 122, were inoculated as checks with a portion of the liver. After the lapse of six months they are alive and well and show no symp- toms of disease. On February 19, 1895, guinea pig No. 332 was also found dead, death being due to inflammation of the bowels, and there was no evidence of tuberculosis. These inoculations made by Dr. Schroeder have confirmed the earlier experiments made by Dr. De Schweinitz, and agree with the following which have been repeated as a check on the first work. They show conclusively that from an originally virulent germ we have succeeded in obtaining an attenuated germ which, even in large doses, is appar- ently harmless to guinea pigs, rabbits, cattle, horses, and monkeys. November 15, 1895, six guinea pigs were inoculated with f c. c. each of an emulsion of an attenuated culture thirty-fourth generation. The pigs weighed as follows: Ouuces. No. 67 9 No. 68 11 No. 60 11 Oiiiict's. No. 70 in No. 71 8l No. 81 - 12 13 November is No. (57 was loiiiul tlead I'rom pneumonia. The other animals exhibited the Ibllowiug condition on subsequent dates : December 13, 1S05. Guinea i)ig. Weight. Ko. C8 No. fi9 Ko. 70 2S'o. 71 No. 81 Ounces. 12 14 13 10 14 Remark.s. Slight local swclliiii;; no enlarged ulands. Do. Do. Slight local .swelling, right inguinal gland en- larged. Slight local swelling; no enlarged glands. December 31, 1895. Guinea pig. Weight. Remarks. No. 68 Oinices. 14 16 15 11 14 Slightly enlarged glands. No. 69 No. 7(1 No 71 Do. No. 81 Slightly enlarged gland. No tuberculosis was evident on autopsy of one of these, showing that the gerin was without pathogenic effect. The animals had steadily gained in weight. Again : These animals were all inoculated on February 5, 180G, with l.ic c. of attenuated tuberculosis culture thirty-seventh generation. On Feb- ruary 13, 189G, the weights of these animals were as follows : Guinea pig- Weight. Ouncen. 9 15i 15 10 12.1 14 10 14 8 14 Remarks. No. 169 Swelling size of a pea at point of incM illation. Do '^ No. 172 No. 174 Do No. 159 Do. No. 170 Large swelling at point of inoculation. Slight swelling at point of inoculation. Do No. 108 No. 171 No. 173 Do No. 164 Slight swelling; pneumonia. Do. No. 166 On February 8 and 10, respectively, guinea pigs No. 167 and 176 were found dead from pneumonia, which they had contracted from exposure to cold. Subsequently guinea pigs Nos. 171, 172, 159, and 171 were 14 found (lead from ])neniii<)iiia. On March 10, 1890, .guinea pig- No. 100, wliicli had been inoculated on February 5, with the attenuated germ was found dead. There were no signs of tubercuk^sis, although the animal had been inoculated over ai month. Tlie pig died from pneu- monia. March 10, 1890, the record of the remaining animals was as follows : Guinea pi J Woifflit. Jleiiiarkis. No. 173 No. 169 No. 168 No. 170 No. 166 Ounces. 15 9 16 10 14 Sliglit soro at point of iuoculatioii. No local lesion. Slight sore at i)oiut of inoculation. Apjtarently well. Do. Guinea pig. Weight. Guinea pig. Weight. Guinea pig. Weight. March 18. 1896: No 168 Ounces. 16 11) 14 14 ]March23, 1896: No.170 No. 168 Ounces. m 164 16' 13J April 7, 1896: No.168 No. 166 No.170 Ounces. 18 No 170 16 No. 173 No. 166 10 No 166 No. 173 No. 173 12 There was no enlargement of the glands indicating the beginning of tuberculosis, and, except a loss of weight in pigs Nos. 170 and 173, which was suspicious, there was no evidence of tuberculosis. These and other cases in which the guinea pigs were inoculated with the attenuated germ, and after one to two years showed no evidence of disease, have proved conclusively the attenuated character of this germ. Although this germ is so attenuated and innocuous to animals, its ability to grow in artificial media is as good and better than ever, and the tuberculin obtained from its culture is as satisfactory as that jjrepared from a more virulent germ. Cows and calves have also been inoculated with this attenuated germ in doses varying from 2 to 500 c. c. at a time without the produc- tion of tuberculosis. Nodules were freciuently formed at the i^oint of inoculation, which seemed, however, to be due to the mechanical action of the germ, and did not jnoduce any evidence of tuberculosis. June 29, 1890. THE EFFECT OF TUBERnXlN INJECTIONS UPON THE MILK OF HEALTHY AND DISEASED COWS. ]\y E. A. ]>K SciiwEiNiTZ, Ph. ]>., .M. ]). Contiiiuiiiii^ the line of experiments given in my report. Bulletin No. 7, Bureau of Animal Industry, upon tlie variation in the amount of fat in the milk before and after the injections of tuberculin, tests were made on different dates upon a healthy cow, 'No. 299, with varying doses of tuberculin, ui)on diseased animals Nos. 145 and 161, and also upon a set of eight difiereut animals taken from the same milch herd — Xos. 185, 186, 187, 189, 194, 195, 222, and 234. The latter had all been condemned by the tuberculin test, and preparatory to their being killed were kept at the station for some days, thus giving an opportunity for testing their milk. There was practically no variation in the fat of the milk from the healthy cows after the tuberculin injection. This agrees with our lirst experiments, and also with some tests made by Dr. Law, reported in Cornell University Bulletin No, 7. Neither was there any alteration when, as is seen from the tests in March on No. 299, large doses, 30 c. c. of tuberculin, were injected. The second and third injec- tion with tuberculin of No. 145 and 161, diseased respectively, caused no appreciable rise of temperature, but there was a decided decrease iu the amount of fat. In the series of January 16, 1895, however, the two animals that showed no rise in temperature failed to show any decrease in the milk fat. When the rise of temperature was noted in the others a marked decrease in fat was also noted. A comparison of the decrease in fat with the extent of the disease, as revealed by autopsy and given me by Dr. Smith, except in case No. 234, a generalized one, does not apparently show anj- relationship. The oldest cases seemed to give the least change in fat — No. 185 — while the newer cases gave the largest variation. The Tables I and II show the quantity and composition of the various samides of milk, and the Tables III and lY the temperature reactions after the tuberculin injec- tion, for the corresponding dates. No. 285, an animal condemned for tuberculosis about a year ago, has been kept at the station since that date. At first she was injected with small doses of tuberculin until she ceased to give a reaction and was again apparently well. The injections of tuberculin were increased in number and (piautity, and on March 20, 1895, the date of the last examination of the milk, the animal received an injection of 100 c. c. 15 16 Previous to that (late she liad received altotiether 505 c. c. of tuberculin. The hist injection caused uo change in the amount of fat or iu the temperature. The variation in fat shoukl, of course, be attributed in part to the fever. But that this is uot the ouly cause is also evident. The varia- tion is uot, judging- from the few tests made, suilicient of itself to prove the presence of tuberculosis, but taken iu conjunction with the rise of temperature might be considered as corroborative evidence. The tuberculin tests were made by Drs. Schroeder and Curtice, while in the milk analyses 1 was assisted by Mr. J. A. Emery. In this connection are appended several charts with curves which show the average variation in temperatures of a number of different animals. These were tabulated in February, 1895, from the reports received from the different States to which tuberculin had been sent. Chart Xo. 1 shows the average temperature of a number of animals, without reference to breeds. Tlie other charts are arranged so that all animals of the same breed are placed together. The diagnoses were not in all cases proved by slaughtering the animals, but in all cases where they were killed the autopsies confirmed the diagnoses. The weights of the animals were taken as the average weight, and the dose of tuberculin was 2 c. c. The Ilolstein cattle showed iippar ently a slightly higher reaction than the others. The temi)erature curve begins at the time of injection, and the first temperatures theu noted are six hours after the injection with tuberculin. The upper line in the charts shows the reaction of the diseased animals, and the lower of healthy ones. In order to make the charts smaller, an average was made of the temperatures of the different animals taken before the injection. From four to eight temperatures were taken of each animal, and the general average thus obtained with a large number of animals is given as the average normal temperature. In the chart giving the curve for the whole number of animals there is a rise of temperature noted after twenty hours. This is due to the Holstein chart, which is included. The late reaction noted in these auimals, which were from different herds, may be due to their larger size. Many objections have been made against the use and reliability of tuberculin as a diagnostic agent, the opposition coming principally from those who are to a great extent unfamiliar with its practical use or who are ouly too ready to condemn a material which, through lack of skill aud knowledge on their part, has perhaps given unsatisfac- tory results. The committee in Paris, composed of Oheveau, Leblanc, Mequin, Nocard, Strauss, Trasbot, aud Weber, reported as follows upon the principal objections to the use of tuberculin: "The use of high temperatures and carbolic acid in the manufacture of tuberculin makes it impossible that the tubeiculin, if properly prepared, should produce disease. It occasionally happens that tuberculin fails to give a reac- tion in diseased animals, but these are very exceptional cases, and occur 17 only sometimes when the animals are very hadly diseased and their con- dition conld be easily recognized, and are not of importance. Occa- sionally, also, apparently healthy animals show a reaction, l)ut when a very thorongh and careful 'autopsy is made evidence of the disease is usually found. In a case of an apparently healthy animal, therefore, one can only say in safety that the examination had not heen sufficiently close to discover the lesions. Again, in cases where there was appar- ently some other disease and the tuberculin injection caused a reaction a careful autopsy has shown the presence of tuberculosis, and that the reaction was due to the latter disease." This disposes of the objection that the tuberculin reaction is not characteristic. The statement that the tuberculin injection causes the disease to spread more rapidly is not warranted by facts, and in many instances the use of tuberculin has apparently caused an iniprovement in the disease. One animal, originally tuberculous, kept at the station of the Bureau of Animal Industry, has received about 3,000 c. c. tuberculin in ditfer- ent injections, extending over a long time. This animal is now well and fat and has entirely recovered from tuberculosis. In 1895 the International Congress for Veterinary Medicine, at Berne, said: "Tuberculin is a most excellent diagnostic material, and can be of the ntmost service in the warfare against tuberculosis." This res- olution was indorsed by the French Academy of Medicine and the use of tuberculin was generally recommended. The satisfactory reports received from the different States to which this Bureau has sent tuberculin are contirmatorj' of the results obtained, and prove that tuberculin is the only effective means at hand to insure a rai^id eradica- tion of tuberculosis in cattle. A table showing the results of the tuberculin injection of more than 50,000 cattle will appear in the next Annual Eeport of the Bureau. February, 1895. 1992— ]S'o. 13 2 18 Table I, — Analyses of milk of cows injected ioith tuherctilin. Date. Quan- tity. Spe- cific grav- ity. Total solids. Sug- ar. Albu- mi- noids. Fat. Ash in milk. 1894. c. c. P.ct. P.ct. P. ct. P.ct. P.ct. Dec. 11 9, 655 1.032 13.65 3.84 3.14 4.48 0.70 Doc. 12 9,655 1.031 14.54 4.17 2.91 6.31 .70 Dec. 13 8,519 1.029 13.38 3.84 2.90 5.10 .62 Dec. 14 9,087 1.029 13.11 4.16 2.86 4.10 .66 Dec. 18 7, 950 1.028 12.74 3.33 2.50 5.49 .60 Dec. 11 2,000 1.018 21.01 5.00 3.2l 15.34 .75 Dec. 12 1,785 1.025 14.68 2.94 3.13 7.73 .75 Dec. 13 3,407 1.026 14. 95 2. 38 3.49 7.16 .72 Dec. 14 3,407 1.027 15.03 3.12 3.51 7.21 .76 Dec. 18 3, 407 1.027 16.26 2.7t 3.75 8.18 .78 Dec. n 450 1.020 18.86 2. 50 3.98 12.56 1.01 Dec. 12 1,025 1.027 12.99 2.63 3.05 5.52 .77 Dec. 13 1,703 1.026 12.77 2.77 2.99 4.09 .74 Dec. 14 1,703 1.026 12. 92 2.94 3.06 4.91 .73 Dec. 18 1,703 1.026 13.73 2.63 3.18 6.35 .81 189.i. Jan. 3 10, 222 1.034 12.61 3.57 2.78 4.26 .73 Jan 4 9,087 1.033 12.34 3.57 2.81 3.30 .76 Jan. 8 10, 222 1.032 12.45 3.57 2.96 3.87 .69 Jan. 9 9,087 1.034 12.58 3.84 2.13 3.86 .67 Jan. 10 9,654 1.033 12.95 3.84 2.51 4.06 .61 Jan. 3 3,407 1. 028 16.97 2.77 3.36 10.14 .86 Jan. 4 3,407 1.030 15.40 3.12 3.05 7.37 .76 Jan. 8 3,691 1.030 15.68 3.12 3.56 7.44 .75 Jan. 9 3,407 1.031 16.74 2.94 3.86 8.35 .73 Jan. 10 3,123 1.028 15.38 3.12 3.58 6.54 .70 Jan. 3 1,703 1.030 17.54 2.94 3.48 9.99 .77 Jan. 4 1,992 1.027 13.27 3.12 2.97 5.77 .83 Jan. 8 2,272 1. 029 14.37 2.94 3.20 6.53 .68 Jan. 9 1,988 1.031 14.63 3.33 3.35 6.52 .70 Jan. 10 1,987 1.031 14.57 3.33 3.28 5.91 .71 Feb. 18 8,234 1.031 11.90 3.33 2.59 3.96 .68 P^eb. 19 8,519 1.033 12.14 3. .57 2.61 3.06 .68 Feb. 20 8,234 1.032 12.33 3.57 2.63 4.07 .68 Mar. 4 8,519 1.031 12.29 3.57 2.72 3.87 .67 Mar. 5 8, 945 1.032 11.99 3.57 2.68 3.46 .08 Mar. 6 1.032 12. iO 3 57 2.69 3.56 .69 Mar. 8 9,087 1.032 11.98 3.57 2.61 3.25 .09 Mar. 19 4,828 1.029 12.84 3.57 3.01 5.14 .67 Mar. 20 5,112 1.029 14. 60 3.57 3.12 6.73 .67 Mar. 21 5,396 1.029 13.59 3.57 3.03 5.81 .66 Total fat acid. (a) 28.39 30.83 36.60 29.21 40.63 38.25 38. 81 27.89 27.21 22. 99 Volatile fat acid. (6) 6.7 5.0 Acidity. (c) 3.9 5.0 4.8 4.8 2.8 5.5 7.5 0.1 5.1 3.3 7.2 7.2 7.5 1.4 1.9 1.9 4.9 5.3 1.4 2.0 4.9 5.3 7.4 7.7 7.0 7.2 8.2 7.4 7.5 5.8 5.2 5.1 e.c. 0. 132 .146 .108 .112 .132 .100 .136 .130 .134 . 114 .072 .072 .102 .110 .090 .140 .150 .168 .156 . 1,54 .100 .156 .122 .138 .138 .130 .132 .124 .130 .124 .128 .132 .150 .154 .156 .160 .156 .106 .096 .098 a Iodine absorption number. b Number of cubic centimeters, - Ba (oH)2, required for 1 gram of fat. N c Kuinber of cubic centimeters, — Ka oH, required for 1 cubic centimeter of n Table II. — Analyses of milk of cows injected with tuherculin. 185. 189. 222. Date. Quan- tity. Specific gravity. Fat. Dat e. Quan- tity. Specific gravity Fat. Date. Quan- tity. Specific gravity. Fat. c. c. c. c. c. c. Jan. 14 5,111 1.029 5.92 Jan. 14 9.655 1.030 4.28 Jan. 14 5,111 1.024 10. 87 Jan. 15 4,260 1.028 5.51 Jan. 15 7,950 1. 028 4.29 Jan. 15 5,111 1.024 8.20 Jan. 16 4,543 1.030 5.50 Jan. 16 7, 983 1.030 3.67 Jan. 16 4,827 1.030 5.09 Jan. 17 4,543 1.030 5.71 Jan. 17 7,607 1.028 4.29 Jan. 17 3,692 1.029 6.53 Jan. 18 4,260 1.029 5.77 Jan. 18 7,667 1.027 3.68 Jan. 18 6,247 1.029 6.12 186. 194. 229. 14 15 Jan. Jan. 14 15 1,420 1,704 1.028 1.025 10.83 11.06 .Tan. Jan. 14 15 2,556 5,679 1.023 1.022 9.03 Jan. 375 1. 028 5.51 • 8.22 Jan. 16 500 1. 022 9.04 Jan. 16 1,998 1.029 6.53 Jan. 16 5,111 1.027 4.91 Jan. 17 568 1. 022 7.19 Jan. 17 2,272 1.030 6.73 Jan. 17 4,260 1.027 6.95 Jan. 18 440 1. 024 6.97 Jan. 18 1,948 L031 5.09 Jan. 18 6,816 1.029 4.28 187. 195. 231. Jan. 14 1,998 1.030 8.77 Jan. 14 5,111 1.024 6.01 Jan. 14 5,111 1.023 8.42 Jan. 15 3,123 1.023 12.32 Jan. 15 1,704 1.027 7.97 Jan. 15 4,543 1. 023 6.36 Jan. 16 3,407 1.028 6.13 Jan. 16 2,272 1.029 4.49 Jan. 16 3,692 1.030 2.44 Jan. 17 3,408 1.030 7.54 Jan. 17 1,998 1.028 8.17 Jan. 17 3,975 1. 027 7.36 Jan. 18 3,408 1.031 6.16 Jan. 18 2, 556 1.030 6.52 Jan. 18 5,111 1.027 5.93 19 Table III. — Temperature reactions of the\cows used in the 7nilk experimenla. 1894. Bi'forc injection. DiciMiibor 11 A/fer injection. DiMiiiilicr 12 (iiijiitid '2 c. c. tulxTculiii at 8 ]>. in. DfciiiilKT 11) 18U5. lief ore injection. JainiMiv After injection. January 8 (in.jci-tt'd "2 c. <•. Uibrrculin at 8 i>. in. January 7) Before injection. February 18. After injection. February 19 (iu.jected 2 c. c. tuborculiu at 9 p. in. February 18) . liefore injection. aiar.b i. After injection. !Marcli f) (iujectt'd ;!() c. c. tuberculin March 4) . 1.00 p.m. 3.00 p.m. 5.00 p.m. 8.00 a.m. 10.00 a.m. 12.00 m. 2.00 p.m. 4.00 p.m. 1.00 p.m. ;).00p. m. 5.00 p.m. 8.00 a.m. 10.00 a. m. 12.00 m. 2.00 p.m. 4.00 p.m. 9.00 a.m. 12.00 m. 4.00 p.m. 9.00 p.m. 8.00 a.m. 9.00 a.m. 10.00 a.m. 11.00 a.m. 12.00 m. 1.00 p.m. 2.00 p.m. 3.00 p.m. 4.00 p.m. 5.00 p.m. 8.30 a. m. 12.00 m. 4.30 p. m. 8.00 a. m. 9.00 a. ra. 10.00 a. m. 12.00 m. 2.00 p.m. 4.00 p.m. 5.00 p. m. No.299. No. 161. No. 145 102.3 102. 101.5 101.0 100.8 101.2 101.0 101.4 101.0 101.0 101. 2 101.5 101.0 100.4 101.4 101.3 101.6 100.6 101. 9 101.2 101.6 102.7 102. 6 100. 2 100.2 101.2 101.5 101.8 101.8 101.4 101.2 100.2 101.2 101.5 102.0 99.2 100. 2 101.2 101.5 101.9 101.3 98.2 99.4 101.0 101.2 100.6 102.0 101.0 102.4 102.6 102.7 1(11.8 101.7 100. 8 101. 101.5 101.6 97.3 98.5 101.4 101.0 102. 5 101.2 101.6 100.6 101.2 101.4 105.2 105. 102. 6 101.5 101.1 20 'J'aiu.k Iv^ — TemperatHrc reactions of the cows used in the milk exjurlmenls. [First test of tubercalous cows.] [Secontl test of tuberculous cows.] 1895. Before injection . January 15 ..•••■.. 4.n0]). in. 9.00 p.m. 102.2 102. 102. 5 101.8 101.8 100. 8 102. 8 102.5 102.0 101.8 101.4 101.8 101.4 101.0 101.6 101.0 100.2 101.4 After injection. January 16 (iu.jectcd 2 c. c. tuberculin 11 p. m. January 15) 8.00 a.m. 101.5 102.0 99. C 102.0 102. G 105.2 104.7 103.2 105.3 8.30 a. ni. 101.8 103.2 101.0 102.0 101.8 105.2 105.2 104.0 105.4 9.00 a. m. 102. 4 103.2 101.2 103.3 102.6 105.2 105.2 104.6 105. 4 9.30 a.m. 102. 2 103.5 101.2 103.2 102. 8 105.3 105. 4 105.4 105.8 10.00 a. 7n. ]02.2 103.6 101.2 103.4 102.8 105.4 105. 3 105. 2 106.0 10.30 a.m. 102.2 103.0 101.6 104.2 103.0 105. 2 106.0 105.5 100. 11.00 a.m. 102.4 103.2 101.5 104.6 104.2 105.1 106.0 105. G 106. 2 11.30 a.m. 102. 6 102.9 101.2 104.2 104.4 105.0 104.6 105.4 106.4 12.00 m. 103. 102.8 103. 2 104.2 104.4 104.8 105. 3 105.0 106.3 12.30 p.m. 103.6 102.6 103.6 104.6 104.3 104.6 10.3. 5 104.8 106.2 1.30 p.m. 103.5 103.0 103.9 104. 8 104.6 104.5 105. 5 105.2 lOG.O 2.00 p.m. 103.4 102.0 104.5 104.8 104.6 104.0 104. 6 104.6 105.6 2.30 p.m. 103.6 101.6 104.6 104.4 104.2 104.0 103. 4 103.5 105.2 3.00 p.m. 102.4 100.0 105.0 100. 102.8 103.4 103.5 102.4 104.6 3.30 p.m. 101.9 99.5 105.0 99.6 102.4 103.0 103.2 102.5 104.2 4.00 p.m. 101.6 99.3 104.6 99.4 102.0 103.2 103. 2 102.6 1C4.0 4.30 p.m. 102.8 99.6 104.5 99.4 102. 103.0 103.2 102.6 104.2 21 -- -^ ~~. -^ ^ --- ^ ^ "^ ^ <:i <:i ?i ^ / s^ V. 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