~ BULLETIN No. 13. U. S. DEPARTMENT OF AGRICULTURE. Ss FE BUREAU OF ANIMAL INDUSTRY. “ A “ ‘euns TUBERCULOSIS INVESTIGATIONS. THE GROWTH OF THE TUBERCULOSIS BACILLUS UPON ACID MEDIA. By E. A. be SCHWkEINITZ and Marton Dorset. FURTHER EXPERIMENTS WITIT AN ATTENUATED TUBERCTLOSIS. BA- ~ CILLUS. By bk. A. pe Scuwerinitz and FE. C. ScuROEDER. THE EFFECT OF TUBERCULIN INJECTIONS UPON THE MILK OF HEALTHY AND DISEASED COWs. By KE. A. pr SCHWEINITZ. Prepared under the direetion of Dr. D. E. SALMON, Chief of the Bureau of Animal Industry. Issned September 19, 1896. WASHINGTON: GOVERNMENT PRINTING OFFICE. ESE . « Ri ee eee Mongree? (lass Book = BULLETIN No. 13. — U. $2 DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. Aq TUBERCULOSIS INVESTIGATIONS. THE GROWTH OF THE TUBERCULOSIS BACILLUS UPON ACID MEDIA. By E. A. pr SCHWEINITZ and MARION DORSET. FURTHER EXPERIMENTS WITH AN ATTENUATED TUBERCULOSIS BA- CILLUS. By E. A. pk SCHWEINITZ and E. C. SCUROEDER. THE EFFECT OF TUBERCULIN INJECTIONS UPON THE MILK OF HEALTHY AND DISEASED COWS. By E. A. pE SCHWEINITZ. Prepared under the direction of Dr. D. E. SALMON, Chiet of the Bureau of Animal Industry. WASHINGTON : GOVERNMENT PRINTING OFFICE. 1896 . »>9 5 4 B ASES.2 3 : a3 > SP ad “Ie \ \ 2 ee, Ad ea bok PL » Vv } a Ost 29 3 3 ps ae? pee ; 7,4 .S.0A#22 96 > ce Arts Ee OF TRANSMITTAL, U.S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Washington, D. C., July 15, 1896, Str: I have the honor to transmit 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 upon 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. EK. A. de Schweinitz, assisted by Drs. Marion Dorset and E. C. 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 Agriculture. Go LETT EROF Sis NEA: U.S. DEPARTMENT OF AGRICULTURE, Washington, D. C., July 1, 1896. Sir: I herewith submit for publication some results of investigations upon the cultivation of the tuberculosis bacillus upon acid media, the innocuous character of an attenuated tuberculosis bacillus, and some further experiments with 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. Respecttuily, HK. A. DE SCHWEINITZ, Chief Biochemie Division. Dr. D. E. SALMON, Chief of Bureau of Animal Indus!ry. CONTENTS. The growth of the tuberculosis bacillus upon acid media. By E. A. de pehweiniz, bh. DS MoD eand! Marion? Dorset, M: D2... ...25-.-.. $22: .--: nmtnodne nome = erate sete cee: eee cn dare toe os oes now ce esate BE 2 CURE IK a SR SSE ae ee ee ee eee Gt EO te Ox PERIM CMSs Acres hes = Se isa oe | Sasha Soon amas sdecce aeua Further experiments with an attenuated tuberculosis bacillus. By. E. A. de pebwemibz, bh. D:, M.D. and: i. C. Schroeder, M.D. V....-...---:-.------- HESPELiMeMGmVAuhra MONK Vurates. ees ae ee = afore sees cisco ase soos eee Resi On Gwined pie IMOCM ALONG ssc. 22 sae seas oases oe eee bse cee The effect of tuberculin injections upon the milk of healthy and diseased cows. Joay LBs. das CRG) Stel ois chu ah ed et ged DSA feel D Jers se ected nas, ie ae ce Analyses of milk of cows injected with tuberculin.........---...---..--.---- Temperature reactions of the cows used in the milk experiments....--...--.- ” 19 Veale 1. 9 a. ILLUSTRATIONS: PLATES. Bacillus tuberculosis upon normal and acid media ......--.....--.---- Bacillus tuberculosis upon acid media containing free hydrochloric acid, and upon artificial mediae-2s-25-222.5222o. oa. ea FIGURES. Average temperature of 3,930 healthy and 1,191 diseased cattle injected with tuberculin... 222 23s: set Sooe eee eee eee Average temperature reaction of Jersey cattle......-.....-----------. Average temperature reaction of Holstein cattle ..-..______-----.---- Average temperature reaction of Durham cattle ---_-........-.--.---- Average temperature reaction of Ayrshire cattle.........--..--------- Average temperature reaction of Guernsey cattle...........---.------ . Average temperature reaction of Grade cattle ..........--..---------- 6 bh bd bo bo or) an =~! TUBERCULOSIS INVESTIGATIONS. THE GROWTH OF THE TUBERCULOSIS BACILLUS UPON ACID MEDIA. By E. A. DE ScuweErniTz, Ph. D., M. D., and Marion Dorset, M. D. INTRODUCTION. In the Philadelphia Medical News of December 8, 1894, one of us (De Schweinitz) published an 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 an acid reaction. This appeared 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 worthy of record. Pawlosky, Ann. de l’Institut Pasteur, 1888 (and Sander) (Archiv fiir Hyg., Bd. 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, 1595, Nos. 4, 5) Lubinski deseribes 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 peptonized 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 find that the tuberculosis bacillus will grow upon acid : : N : ; beef broth which requires 21.6 ¢. ¢. 10 sodium hydrate solution to neu- tralize every 100 c. c. of the media. It will also grow upon acid arti- We : : , A aes Ge as ficial media every 100 ¢. c. of which requires 45 ¢. c. of 10 sodium hydrate solution for neutralization. This acidity in some instances was due to the acids in 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 4 pi 8 : ; N and to the acid artificial media, so that from 1 ¢. ¢. to 3 ¢. e. | hydro- _ chloric acid have been contained in every 100 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 congo red or phloroglucin and vanillin. The congo-red 7 8 test was not satisfactory in the beef broth, the albuminoids and pos- sibly organie acids present evidently interfering with the 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. of cul- ture 12.5 c. c. to 15 ¢. ¢. Es sodium hydrate. Lubinski 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 filaments, 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 ecul- 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 morphological changes are somewhat differ- ent. Instead of a thread-like growth or chain growth we 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 upon our acid media can be seen from the accompanying plates. PI. I, fig. 1, shows the bacillus grown upon glycerin beef broth for four months; Neisser’s spore stain. P1. 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 bespore 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 fiir Bak. und Parasitenkunde, Bd. X VII, Abth. 1, No. 23) describes a germ which gives Neisser’s spore reaction and in that particular evidently corresponds to the ones we have pictured. Bul. 13, Bureau of Animal Industry. A \ ‘ = / l : * Ly - NS = ‘4 Bf uate Weg Se aD, ’ Fie. 1.—Seventh generation in glycerine beef broth. Neisser’s spore stain. (> 2000.) —_ ov ‘ee 2 Fig. 2. —Fortieth generation, acid media. Neisser’s spore stain. (>< 2000.) Haines, del. BACILLUS TUBERCULOSIS UPON NEUTRAL AND ACID MEDIA. Bul 13, Bureau of Animal Industry PLATE II. ae aoe #7 ay ~- “ Te { : / eo eae g . Fic. 1.—Culture containing free hydrochioric acid. Neisser’s spore stain. ( 2000.) =e —_ es \ ( the Gen Fic. 2.—Artificial culture. Neisser’s spore stain. (x 2000.) Haines, del. BACILLUS TUBERCULOSIS UPON ACID MEDIA CONTAINING FREE HYDROCHLORIC ACID, AND UPON ARTIFICIAL MEDIA. 9 Pl. I, fig. 1, shows the germ grown upon acid media containing free hydrochloric acid. PI. U, fig. 2, is a drawing of the tuberculosis germ grown on arti- ficial media. Wedo 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 process of sterilization. A quantitative check of the amount of acid was made before and after this process. First experiment.—The media used for this experiment was alkaline ; reas N : and required for neutralization 1c. ¢. 5, hydrochloric acid for every 50 ¢. ¢. of the media. NaOH were added. N a. To each of 2 flasks 50 e. c. each, 5 e. ¢. 10° N “10 NaOH were added. N ce. To each of 2 flasks 50 c. ¢. each, 1 c. e. 4, HCl were added. b. To each of 2 flasks 50 ¢. ¢. each, 20 ¢. ¢ 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 ¢, while ) showed no growth at all. Second experiment.—The media used required for neutralization 8,4 N ; C. C. 76) sodium hydrate solution for every 50 c. c. N a. To each of 2 flasks 50 ec. e. were added 8.4 e. e. 10° NaOH. b. To each of-2 flasks 50 ec. c. were added 1.3 ¢. c. 5, HCl. a flasks were neutral and b flasks acid. The two sets were inocu- lated from the same culture (A 15). Within two weeks the germs in both flasks were growing well, a perhaps a trifle better than b. At the end of seven weeks one flask, a, was titrated and required for neutral- ae N : ization 6.3 ¢. ¢. 10° NaOH. This flask had been neutral when inoculated. : N sere. . b flasks required 22.5 ¢. ¢. 10° NaOH for neutralization. The increase Sag : N in acid in @ flasks is represented, therefore, by 6.3 ¢. ¢. 10° NaOHd, and in the b flasks by 7.5 ¢. ¢. NaOH. There was apparently a larger amount of the acid substance formed in the acid flasks than in those which were neutral at the time of inoculation. Third experiment.—The media used required for neutralization 6.6 N C. C. 79) NaOH for every 100 ¢. ¢. of liquid. 10 r) N a. To each of 2 flasks 100 ¢. ¢.,1 ¢. e. 5, HCl was added. N b. To each of 2 flasks 100 ¢. ¢.,2 ¢. ©. 97 HCl were added. ; N c. To each of 2 flasks 100 e. ¢.,3 ¢. e. 5) , 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 cuitures, the most acid ones seeming to contain as good a growth as the others. Every 100 ¢. c. of the a media N had required 11.6 ¢@. ¢. 10° NaOH. Every 100 c. c. of the b media had required 16.6 e. e. NaOH. N 10” - ; ; : N Every 100 ¢. c. of the ¢ media had required 21.6 ¢. ¢. 10” NaOH for neutralization. Fourth experiment.—The media used in this experiment was the artificial media described by one of us—De Schweinitz—in 1892, and it seal INS se ahe 3 ee required 45 e. ¢. 10° NaOH for every 100 ¢. c. for neutralization. «100 c. e. one flask of the media as noted. b 100 ¢. ce. one flask of the media as noted with the addition of 2 ¢. ec. N : |» 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 germ. It may bea 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 produced 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. Marcu 14, 1896. FURTHER EXPERIMENTS WITH AN ATTENUATED TUBERCULOSIS BACILLUS. By E. A. DE ScHWEINITZ, Ph. D., M. D., and KE. C. SCHROEDER, M. D. V. In the Medical News, December 8, 1894, one of us (EK. A. de Schwei- nitz) 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 4c. ¢. 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 $ ¢. ¢. of tuberculosis eaten’ thirty-second generation was repeated, and again on January 20, 1896, 2 ¢. c. of tubercle culture thirty-third 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 Inngs 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 RESULTS OF GUINEA PIG INOCULATIONS. The guinea pig inoculations made by one of us (Schroeder) with this attenuated germ to test its virulence were as follows: November 24, 1894, Nos, 280, 281, 282, and 283 received each an abdominal injection enbotancoaely of $c. c. tubercle culture twenty-third generation. On January 25, 1895, each pig again received 1 e. ¢. tubercle culture twenty-sixth penevation! October 7, 1895, guinea pig No. 281 was found dead. A small dry, cheesy cane at ae 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, 1896, 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, 56, 57, 58, and 59 were inoculated on October 2, 1895, subeutaneously with 1 ¢. ¢. 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. 56, 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 § ¢. 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 jada ween 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- eutly harmless to guinea pigs, rabbits, cattle, horses, and monkeys. November 15, 1895, six guinea pigs were inoculated with 3 ¢. ¢. each of an emulsion of an attenuated culture thirty-fourth generation. The pigs weighed as follows: Ounces. Ounces. INOS O(a Seo eo eee Pre Ow ret Ap aan sere 9 No. 20.23 eS Bae eee 114 INO GS Sie ee eee es Se eee ED | INO: TIS a ee 8} NOD G9 eceeaa tere Soeeee eae eee ee 11 No. OL. cose a seet one clan eee eee 12 13 o November 18 No. 67 was found dead from pneumonia. The other animals exhibited the following condition on subsequent dates: December 13, 18985, Guinea pig. | Weight. Remarks. | Ounces. IU) LS oo Sooo abe ce cS nb Boee SSE OnOS Se eee OEse 2 | Slight local swelling; no enlarged glands. (lb) Gl eee eet te Seen e See pApOUSOroS 14 Do. , “Sith, Te poo aS ie ee ene er 13) | Des wl ile SS SS Sie eee ne soe Soe 10 | Slight local swelling; right inguinal gland en- larged. INORG. ccs sce. oon 2 ocohboctossesaubEcassse 14 Slight local swelling; no enlarged glands. December 31, 1895. Guinea pig. Weight. | Remarks. Ounces. 14 | Slightly enlarged glands. 16 | No enlarged glands. 15 Do. 11 Do. 14 Slightly enlarged gland. No tuberculosis was evident on autopsy of one of these, showing that the germ was without pathogenic effect. The animals had steadily gained in weight. Again: | | Guinea pig. Weight. | Guinea pig. Weight. Guinea pig. | Weight. Ounces. Ounces. |, Ounces. INO LOUr ars <)cs~ -nsesec Ta | sen Co So eras ieee NASW ON OD ATA pesto oot oo: 15 i Cy 11 3 | es cee be ee RIAs DUS PIN OnllyGies cscs eee ea } 14 PO GHe n= ca--aock tc. AAO NOmnies=2o-cces.2265.- 13H || aN 6!) Son Soha 10 1D Li eos 10 Nomis js 3sccs2 - Soeee 1G No vlGsee ee S22: soacad- 10 These animals were all inoculated on February 5, 1896, with 14 c.¢. of attenuated tuberculosis culture thirty-seventh generation. On Feb- ruary 13, 1896, the weights of these animals were as follows: | % Guinea pig. | Weight. Remarks. | | Ounces. 1D Tn. ae a ee 9 | Swelling size of a pea at point of inoculation. "1 fkb Oe eee 158 Do. INith dt aS eeeee een AGE SSAe cena sea Sse 15 Do. 1h 12) ee Bae eee 10 Do. LUTE UT el ony aS © Sa a 124 | Large swelling at point of inoculation. LU OE eee ee ee 14 | Slight swelling at point of inoculation. [ANCE WA RAE oS RE Naat ae ea Oe 10 Do. PSM eRe ae ieee ane Kew cf Sanne 14 Do. DVO RMOA ee erence seco reulosseusseessesees uc 8 | Slight swelling; pneumonia. inh. Se Ce eee a a 14 Do, 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. 174, 172, 159, and 171 were 14 found dead from pneumonia. On March 16, 1896, guinea pig No. 169, which had been inoculated on February 5, with the attenuated germ was found dead. There were no signs of tuberculosis, although the animal had been inoculated over a month. The pig died from pneu- monia. March 10, 1896, the record of the remaining animals was as follows: Guinea pig. | Weight. Remarks. | Ounces. | INO LTB naece = ees ete anaes eeneeeee | 15 | Slight sore at point of inoculation. ING. 160 set eadeseS aceon ete seems Smates 9 | No local lesion. INOS 16885.5 223 25d edeneeeciae cee on emer 16 | Slight sore at point of inoculation. INOMO reece ae nae ee an eee eerie 10 | Apparently well. INO: AG62 22 cccie estes cceemacen ene coer te ao 14 Do. Sa otha Fy ea ts en ee Ss ea Guinea pig. | Weight. | Guinea pig. Weight. Guinea pig. Weight. | ; | | | March 18, 1896: Ounces. | March 23, 1896: | Ounces. | April 7, 1896: Ounces. No. 168 16 | INOMIT0 eae ccacee 104 INOSWESee et eeacee No. 170 | 10 | INOs68) oe eee 163 INOj166 = See 16 No. 173 -- 14 | IN@s WUD <2 cacceccee 16 INOS eee 10 No. 166 14 NOM LT Sieneacesee = 13% | INO oeseeereeee ee 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 prepared 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 frequently formed at the point of inoculation, which seemed, however, to be due to the mechanical action of the germ, and did not produce any evidence of tuberculosis. JUNE 29, 1896. THE EFFECT OF TUBERCULIN INJECTIONS UPON THE MILK OF HEALTHY AND DISEASED COWS, By E. A. DE SCHWEINITZ, Ph. D., M. D. Continuing the line of experiments given in my report, Bulletin No. 7, Bureau of Animal Industry, upon the 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, upon diseased animals Nos. 145 and 161, and also upon a set of eight different animals taken from the same milch herd—Nos. 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 first 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 in 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 any 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 samples of milk, and the Tables Il] and LV 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 quantity, and on March 20, 1895, the date of the last examination of the milk, the animal received an injection of 100 c. ¢. 15 16 Previous to that date she had received altogether 565 ¢. c. of tuberculin. The last injection caused no change in the amount of fat or in the temperature. The variation in fat should, of course, be attributed in part to the fever. But that this is not the only cause is also evident. The varia- tion is not, judging from the few tests made, sufficient of itself to prove the presence of tuberculosis, but taken in 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 I 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 No. 1 shows the average temperature of a number of animals, without reference to breeds. The 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 ¢.¢. The Holstein cattle showed appar ently a slightly higher reaction than the others. The temperature curve begins at the time of injection, and the first temperatures the! 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 thes animals, 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 only too ready to condemn a material which, through lack of skill and knowledge on their part, has perhaps given unsatisfae- tory results. The committee in Paris, composed of Cheveau, Leblanc, Mequin, Nocard, Strauss, Trasbot, and Weber, reported as follows upon the principal objections to the use of tuberculin: “The use of high temperatures and carbolic acid in the hanufacture of tuberculin makes it impossible that the tuberculin, 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 badly diseased and their con- dition could be easily recognized, and are not of importance. Occa- sionally, also, apparently healthy animals show a reaction, but when a very thorough 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 been 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 improvement in the disease. One animal, originally tuberculous, kept at the station of the Bureau of Animal Industry, has received about 3,000 ¢. ¢. tuberculin in differ- 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 utmost service in the warfare against tuberculosis.” This res- olution was indorsed by the French Academy of Medicine and the use of tubereulin was generally recommended. The satisfactory reports received from the different States to which this Bureau has sent tuberculin are confirmatory of the results obtained, and prove that tuberculin is the only effective means at hand to insure a rapid 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 Report of the Bureau. FEBRUARY, 1895. 1992—No. 13 2 J 18 TABLE I1.— Analyses of milk of cows injected with tuberculin. ; | Spe- . a . No.of} oases ‘ Albu- | Ash Total | Volatile eee ani- | Date ea ale ee NRE: Ini- Fat. | in | fat acid. | fat acid. | Acidity mal. ve Bie i eck mearPRSLOYoytal sp milk. (a) (Db) (¢) 1894. €: Cs IPRCGAM Pe ECes || eatin eelCLeml eeenee Pet (Oates CC. 299 | Dee. 11 9, 655 1. 082 13. 65 | 3.84 3.14 AAR sNOnOslcoete ness 6.7 0. 132 Dec. 12 9, 655 1. 031 14,54 | 4.17 2. 91 6.31 S{Ulh | Seeppesacs 5.0 . 146 Dee. 13 8, 519 1. 029 13.38 | 3.84 2.90 5.10 SOON iccte isle roe 3.9 - 108 Dec. 14 9, 087 1. 029 13.11.| 4.16 2.86 | 4.10 BOBS xisccms ees 6.6 . 112 Dec. 18 7, 950 1. 028 12. 74 | 3.33 2.50 5.49 Fae ee eS AST.) «132 145 | Dec. 11 2,000 | 1.018 21.61 | 5.00 3.21 | 15. 34 «BO Nsisses ccm 3.5 | - 100 | Dee. 12 1, 785 1. 025 14.68 | 2.94 3.13 | 7.73 ait AReeroactec 3.9 | . 136 Dec. 13 3,407 1. 026 14.95 | 2.38 3.49 | 7.16 OT onl poee eee cer 5.0 | . 130 Dec. 14 | 38,407 1. 027 15. 03 | 3.12 3.51 | 7.21 COt eee 4.8 | ~ 184 Dec. 18 | 3,407 1. 027 16.26 | 2.7 ABiBy | we ales wh Ot | seiee ems 4.8 | 114 161 | Dec. 11 450 1. 020 18. 86 | 2.50 8.98 1) LeO0n| ele Oly ee ee eee 2.8 | 072 | Dec. 12 1,025 | 1.027 12.99 | 2. 63 3. 05 5. 52 PY Cal ees P aS 5.5 . 072 | Dec. 13 1,703 | 1.026 12.77 | 2.97 2.99 | 4.09 Pa fal |e ear ea 1.5 . 102 Dec. 14 1,703 | 1.026 12. 92 | 2.94 3. 06 4.91 Sie. eee es 6.1 .- 110 Dec. 18 1,703 | 1.026 13.73 | 2.63 3.15 6.35 Atl Ee Re Te 5.1 . 090 1895. 299 | Jan. 3 | 10, 222 1. 084 A2nOL We pw 2.78 4, 26 LOR seein 3.3 . 140 Tank 45 T9NOST al MeO8S ||) MONS 4a! tS 57 ee ONS e585 0 eT 6 | eee ome | a acl Sei Jan. 8 | 10, 222 1. 082 12.45 | 3.57 2.96 3.87 ACB Sees ee 1.2 - 168 Jan. 9] 9,087 1. 084 12.58 | 3.84 2.13 3. 86 sOTS | Pecmhecitecs Eig . 156 Jan. 10 | 9,654 1, 033 12.95 | 3.84 2.51 4.06 SE | eee ee ee WD - 154 145 | Jan. 3 3,407 | 1.028 16.97 | 2.77 3.36 | 10,14 Po See ee S 1.4 - 100 Jan. 4 3, 407 1.030 15.40 | 3.12 3. 05 7.37 ANAS ese 1.9 - 156 Jan. 8 3, 691 1. 030 15. 68 | 3.32 3. 56 7.44 BGS Seatac 1.9 . 122 |can., 9! | i407 1.031 16.74 | 2.94 3.86 | 8.35 SUM es oot ests 4.9 - 138 | Jan. 10 3,123 |, 1.028 } 15.38 | 3.12 3. 58 6. 54 AAU eepaseres 5.3 - 138 161 Jan. 3 | 1,703 1. 030 17. 54 | 2.94 3.48 | 9.99 wtih i saeapas stcree 1.4 130 | Jan. 4 1, 992 1, 027 13.27 | 3.12 2.97 | 5.77 ZEB Rechte eee 2.0 -o2 Jan. 8 2, 212 1.029 14. 37 | 2.94 3. 20 6.53 268: \oosee 2 54| eee - 124 | Jan. 9 1, 988 1. 031 14. 63 } 3.33 3.35 | 6.52 AW AU Peon Sk Soo 4.9 |*s .130 Jan. 10 1, 987 1. 031 14.57 | 3.33 3.28 | 5.91 Ait Oe ee 5.3 124 299 | Feb. 18 8, 234 1. 031 11.90 | 3.33 2.59 | 3.96 . 68 28. 39 7.4 - 128 Feb. 19 | 8,519 1.033 12.14 | 3.57 2. 61 3. 66 . 68 30. 83 Leth - 132 Feb. 20 8,234 | 1.032 12. 33 | 3.57 2. 63 4.07 . 68 36. 60 7.0 - 150 299 | Mar. 4] 8,519 1.031 | 12.29 | 3.57 ahaa) (Ned . 67 29. 21 tee ~ 154 Mar. 5| 8,945 150325) L199) 3557 2. 68 3.46 . 68 40. 63 8.2 - 156 Mae eNG Sec cre 1. 032 12.20 | 3.57 2.69 | 3.56 . 69 38. 25 7.4 - 160 Mar. 8) 9,087 1. 032 11.98 | 3.57 2. 61 3. 25 . 69 38. 81 (ee 156 285 | Mar. 19 | 4,828 1. 029 12. 84 | 3.57 3. OL 5.14 . 67 27. 89 5.8 - 106 Mar. 20 5, 112 1. 029 14.60 | 3.57 3.12 6. 73 . 67 Zeal 5.2 . 096 Mar, 21 5, 396 1.029 | 13.59 | 3.57 3.03 5.81 . 66 22. 99 5.1 . 098 a Iodine absorption number. b Number of cubic centimeters, a Ba (0H)», required for 1 gram of fat. ec Number of cubic centimeters, i Na oH, required for 1 cubic centimeter of milk. TABLE Ii.—Analyses of milk of cows injected with tuberculin. 185. } 189. 222, uan- |Specific) | Quan- |Specific uan- [Specific Date ee gravity. Fat. || Date tity. |gravity Fat Date pies ee Fat. (ey OS | c. C. Cc. C. CUE) cee © Se era DE 1, 029 5.92 || Jan. 14 9,655 |; 1.080 | 4.28 || Jan. 14 tap, LUG 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 1.080 5.09 Jan. 17 | 4,548 1. 030 5.71 || Jan. 17 7, 667 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. 1 SAT. MUA Ree. socio: tsrevseciernell bats ~tains= Jan. 14 1,420 | 1.028} 10.83 |} Jan. 14 | 2,556 1. 023 9.03 dan. 15 375 1.028 | 5.51 || Jan. 15 1, 704 1.025 | 11.06 |j Jan. 15 5, 679 1.022 | - 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 | 1.031} 5.09 || Jan. 18} 6,816 1, 029 4, 28 187. 195. 234. Jan. 14 1, 998 1.030 | 8.77 || Jan. 14 | 5, 111 1. 024 6.01 || Jan. 14 oy 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 (eat) || aiehon aly’ 1, 998 1. 028 8.17 || Jan. 17 8,975 1. 027 7. 36 Jan. 18 3, 408 1.081 6.16 || Jan. 18 2, 556 1. 030 6.52 || Jan. 18 5,111 1. 027 5. 93 - ey TaBLE III.—Temperature reactions of the-cows used in the milk experiments. | Date. | Time. No. 299.| No. 161. No. 145. 1894. | Before injection. | ote 6 5 I DYeTAS TN endl U bee 3" Bane Se ASS Se ee: See ae eee ee 1.00 p.m. 102.3 | 101.3 101.6 | 3.00p.m.| 102.0] 98.2 97.3 5.00p.m. | 101.5 99.4 98.5 After injection. H | | December 12 (injected 2 ¢. c. tuberculin at 8 p.m. December 11)-.-) 8.00 a.m. 101.0 | 101.0) 101.4 : 10.00 a.m. 100. 8 101. 2 101.0 12.00 m. 101, 2 100. 6 102.5 2.00p.m. | 101.0| 102.0] 101.2 4.00 p.m. 101.4 101.0 | 101.6 1895. 2 Before injection. JipitGhe) [lee neetodsec ipa snes ssa sesso sees ose ceodensocaotcpasee cor 1.00 p.m. 101.0] 102.4 100.6 3.00p.m.} 101.0) 102.6 101.2 5.00 p.m. 101.2 | 102.7 101.4 After injection. | | January 8 (injected 2 ¢.c. tuberculin at 8 p.m. January 7)--.----- 8.00 a.m. 101.5 101.8 105. 2 10.00 a. m. 101.0 101.7 105. 0 12.00 m. 100. 4 100. 8 102. 6 2.00p.m.| 101.4] 101.0 101.5 ~ 4.00 p.m. 101.3 101.5 101.1 Before injection. February 18... -- Loe SBE dG OAS bee UE Sees yp eS Bad Seo See See (Or OO laeanes | el Oln Gleam = eee eae | 12.00 m. T0086); a2 eee enfasenscss 4.00 p.m. Uh ee sl ere LOO ere cai LON 2 areal oh ofa east After injection. February 19 (injected 2 c.c. tuberculin at 9 p.m. February 18)..-) 8.00 a.m. (ne anes BAbeacee 9.00 a.m. aU al Pee es | ee, 10.00 a.m. OZAGH SRS s see ee mata cta 11.00 a.m. OOF 2 eevee | eee ere 12.00 m. MOOS 2a Rete otter Io ene |i) UEP Se aes eee cco SOU series CLOUD Rem one srr oreonoc SOO josi! GCN RS epee loseaccce £200ipemi | TOLSi|Seec. SBeeo6ce 5.00 p.m. LO Aa Ee «apt PS aeeou ae Before injection. | WWieighiide Seer oo Sees cers i oe Saad Ses eee ee ee ee OUD Etisal) a NS PA Sis Seats eee oe 12.00. ( HUNbPs | paece Sse ees ZTE yest || S0VIGO al egal eee ee After injection. | i} March 5 (injected 30 c.c. tuberculin March 4)...........--------. CeO wie || ADey Se ooeme feseioe. 9.00 a.m. OZ ON eerste eaaaacce 10.00 a.m. CHT oe asobe eeeeouce 12.00 m. MOOS DR eee Seecsere DOO peme he lOls seo oes ae Jeeoweate 22 00ipaa. Ole bese 22 8 Weaaemen’. 500m ets | LOO) ESS oor | eee 20 TABLE IV.—Temperature reactions of the cows used in the milk experiments. [First test of tuberenlous cows.] No. | No. a ae No. No. No. | No. Date. Time. 185. || 186) | 187, ale 189. |) 1945") 195. ||| 2e5 aon ead ee ea — —| — | = ——s 1894. | Before injection. eo} °o is} ° io} oO 9) oO ° December’? ----=2--<--- 9.00 p.m. | 102.0 | 100.5 | 100.2 | 101.5 | 101.2 | 102.5.) 102.2 | 101.3 | 103.9 After injection. | December 8 (injected 24 | ec. ¢. tuberculin 11 p. m. | December 7). ce-ch. 8.00 p.m. | 102.4 | 101.2 | 101.3 | 102.0 | 102.0 | 105.8 | 102.6 | 105.4 | 103.0 9.00 p.m. | 103.1 | 101.4 100.3 | 101.8 | 103.8 | 107.0 |.103.6 | 104.8 | 103.6 10.00 p. m. 105.3 | 103.2 | 102.4 | 104.8 | 104.0 | 107.2 | 104.6 | 103.4 | 103.8 11.00 p.m. | 105.6 | 105.6 | 102.1 | 105.8 | 103.8 | 107.2 | 106.4 | 106.2 | 104.8 12.00 m. 106.6 | 106.0 | 101.5 | 106.6 | 104.3 | 107.4 | 106.2 | 106.9 | 104.5 2.00 a.m. | 106.0 | 105.6 | 99.7 | 106.4 | 106.1 | 106.7 | 106.5 | 106.5 | 105.2 4.00 a.m. | 105.4 | 105.1 | 104.7 | 105.6 | 105.2 | 105.3 | 105.0 | 105.4 | 105.7 8.00 a.m. | 103.0 | 106.2 | 104.8 | 105.6 | 102.0 | 104.2 | 105.2 | 104.4 | 104.7 10.06 a.m. | 101.0 | 100.5 | 101.0 | 100.5 | 101.0 | 101.1 | 101.0 | 100.4 | 102.6 [Second test of tuberculous cows. ] 1895 Before injection. January 15 ....... e«e---| 4.00 p.m. | 102.2 | 102.5 | 101.8 | 102.8 | 102.0 | 101.4 | 101.4 | 101.6 | 100.2 9.00 p.m 102.0 | 101.8 | 100.8 | 102.5 | 101.8 | 101.8 | 101.0 | 101.0 | 101.4 After injection. January 16 (injected 2 c. ¢. tuberculin 11 p.m. January 15)......-...- | 8.00 a.m. | 101.5 | 102.0 99.6 | 102.0 | 102.6 | 105.2 | 104:7 | 103.2 | 105.3 8.30 a.m. | 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.m. | 102.2 | 103.6 | 101.2 | 108.4 | 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 | 106.0 11.00 a.m. | 102.4 | 103.2 | 101.5 | 104.6 | 104.2 | 105.1 | 106.0 | 105.6 | 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. 103.0 | 102.8 | 103.2 | 104.2 | 104.4 | 104.8 | 105.3 | 105.6 | 106.3 12.30 p.m. | 103.6 | 102.6 | 103.6 | 104.6 | 104.3 | 104.6 | 105.5 | 104.8 | 106.2 1.30 p.m. | 103.5 | 103.0 | 103.9 | 104.8 | 104.6 | 104.5 | 105.5 | 105.2 | 106.0 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 | 100.0 | 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 | 104.0 4.30 p.m. | 102.8 99.6 | 104.5 99.4 | 102.0 | 103.0 | 103.2 | 102.6 | 104.2 21 Fria. 1.—Chart showing average temperature of 3,980 healthy and 1,191 diseased cattle injected with tuberculin. (a) [Average normal temperature of 1,191 tuberculous cattle before injection, 101.69 F. Average normal temperature of HOURS AFTER /NJECT/ON. aes Sa a Vv, a Tabulated in February, 1895. b Temperature at time of injection. 920 healthy cattle before injection, 101.5° F.] 22 ‘morjoalur JO amt, ye oanguaidduey, 7 eget ‘Aaenaqa,y UL poyepnqey, v “a - a eer freer 2 sae ted NOLLIFILNI YILSIV SYNOH [af of LOL ‘woroolur oroyoq soo Ly way [6g JO ANZwAsdUI9} [VULION OSVABAY “VT of TOL ‘Worjoofur o«ojoq SAroo SnopTNo1aqny Tez JO On jwsEduta} [VULIOM ODVIIAY | () *wypnosagne ypu pajoalur shasuap liyppay GET pun snojpnoadaqny 8G fshossap apoab hyzyway Gog puv snopnawaqny [ZL 7 F & oa i, i ‘ 4 ~~ 2 J i . a > a ae * 4 —< ye ee ‘ 4 a P : ay — i 7 - z - fe —_ ‘ t ; 2 * . Pee - _* ONGRESS LIBRARY OF © |