Tuberculosis investigations

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Material Information

Title:
Tuberculosis investigations
Series Title:
Bulletin / U.S. Department of Agriculture, Bureau of Animal Industry ;
Uncontrolled:
Growth of the tuberculosis bacillus upon acid media
Further experiments with an attenuated tuberculosis bacillus
Effect of tuberculin injections upon the milk of healthy and diseased cows
Physical Description:
27 p., 2 leaves of plates : ill. ; 23 cm.
Language:
English
Creator:
Dorset, M ( Marion )
Schroeder, E. C ( Ernest Charles ), 1865-1928
De Schweinitz, E. A ( Emil Alexander ), 1864-1904
Salmon, D. E
Publisher:
U.S. Dept. of Agriculture, Bureau of Animal Industry
Place of Publication:
Washington, D.C
Publication Date:

Subjects

Subjects / Keywords:
Tuberculosis   ( lcsh )
Tuberculosis in animals   ( lcsh )
Genre:
federal government publication   ( marcgt )
non-fiction   ( marcgt )

Notes

Additional Physical Form:
Also available in electronic format.
Statement of Responsibility:
prepared under the direction of D.E. Salmon.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 029609942
oclc - 22533807
System ID:
AA00025983:00001

Full Text








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0100I8 INVESTIO ATIONS.



THE TUBERCULOSIS BACILLUiS UPON ACID MEDIA.
ByE. A. DE fCHWEINITZ anld MARION DQRSET..
METS WVITH A:'N ATTENJJATE D TUBERCULOSIS9 BA-
By V.. A. Dm SCH~WEINITz and E. C. ScHRONDER..
9RC.TJLINJNJECTIONS UPON THE MlILK OF HEFA:LTHY,
,By E. A. DE, SCHWEINITZ.


aredl Under the direction of
*Ol D. -E. SALMON.



16<1ed September 19, 1896.
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U. S. DEPARTMENT OF AGRICULTURE.
BUREAU OF ANIMAL INDUSTRY.






TUBERCULOSIS INVESTIGATIONS.




E GROWTi OF THE TUBERCULOSIS BACILLUS UPON ACID MEDIA.
By E. A. DE SCIIWIINrrT and MARIN DORSET.
FUTER EXPERIMENTS WITH AN ATTENUATED TUBERCULOSIS BA-
LUS.
By E. A. .DE SCIIWvISNIT and E. C. Sc(,IOElI)ER.
CIET OF TUBERCULIN INJECTIONS UPON TIIE MILK ()F IIEALTHY
ND DISEASED COWS.
By E, A. A.E SCI> WENITZ.



Prearpred uider the direction of
Dr. D-. E. SALMON
+hie*' o' the b BllreauL o>1' Laitirnu.l In(tlust ry.



-. ., T< Issu2iied Sc<+ltembri 19, 1 96i.
















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LETTER OF TRANSMITTAL.



U. S. DEPARTMENT OF AGRICULTURE,
BUREAU OF ANIMAL INDUSTRY,
Washington, D. C., July 15, 1896.
i: I have the honor to transmit herewith, and to recommend for
pbli n as a bulletin of this Bureau, the manuscript containing a
rerof investigations upon the cultivation of the tuberculosis bacil-
s un aid media, and of some further experiments with reference to
t of tuberclin upon the milk of cows.
experiments have been conducted under the immediate super-
visin of Dr. E. A. de Schweinitz, assisted by Drs. Marion Dorset and
E. Schroer, and the publication of the results obtained will furnish
Scvit and desirable method of preserving and distributing the
S of scientific investigations in this important part of the work
of this Bureau.
Very respectfully,
1). E. SALMON,
Chief of Bureau.
111. J. STERLING MORTON,
Se retar of Agriculture.


8f/Is/IH il 4fHPittl'Wf





















LETTER OF SUBM ITTAL.



IT. 8. I)EPARTMENT OF AGRICULTURE
Washington, D. C., Jiy 1, 1896.
Smi: 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.
Respectfilly,
E. A. DE SCHWEINITZ,
Chief Biochemic Divisi n.
])r. 1). E. SALMON,
Chief of Bureau of" Anal Indius'ry..








I;,





























CONTENTS.


Tnge.
The growth of the tuberculosis bacillus upon acid medlia. By E. A. do
Schweinitz, Ph. D., M. D., and Marion DIorset, 1. D .......-.............. 7
Introduction ....................... ... ... ...... ...... ...... .... 7
Observations ....... ...... ............-...... ...... ...... .. ..... ..... 8
Details of experiments .......... .... ...... .... .......- .......-. .. -.. .. 9
Further experiments with an attenuated tuberculosis bacillus. By. A. A. d
Sehweinitz, Ph. D., M. D., and E. C. Schroeder, 1). ...................--------. 11
Experiment with a monkey ......---...............................--..... 11
Results of guinea pig inorlations....................................... 12
The ffe-t of tuberculin injections upon the milk of healthy and diseased cows.
By E. A. de Schwcinitz, Ph. 1)., M. ) .................. ..................--- 15
Aayses of milk of cows injected with tuberulin .........................--- 18
Tunperaturo reactions of the cows used in the milk experimients ....-..-.. 19
























ILLUSTRATIONS.




PLATES.

P1. 1. Bacillus tuberculosis upon normal and acid media ....--...------- .... 8
2. Bacillus tuberculosis upon acid media containing free hydrochloric acid,
and upon artificial media .....................----....-- ..............- 9

FIGURES.

Fig. 1. Average temperature of 3,930 healthy and 1,191 diseased cattle injected
with tuberculin.................................................... 21
2. Average temperature reaction of Jersey cattle......-.................. 2
3. Average temperature reaction of Holstein cattle .....--.-- .... ..... 23
4. Average temperature reaction of Durham cattle ...................... 24
5. Average temperature reaction of Ayrshire cattle...................... 25
6. Average temperature reaction of Guernsey cattle -........ ........- -26
7. Average temperature reaction of Gradee cattle ....................... 27
6















.I











TUBERCULOSIS INVESTIGATIONS.



THE GROWTH OF THE TBERCULOSIS BACILLUS UPON ACID MEDIA,
By E. A. DE SCHWEINITZ, Ph. D., M. D., and MARION DORSET, M. D.
INTRODUCTION.

In the Philadelphia Medical News of December 8, 1894, one of us
(De chweinitz) published an article upon the attenuated bacillus tuber-
ulosis ad 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 fir
Hyg., ed. XVI, p. 238) have shown that vegetable broths may be used
r the cultivation of the bacillus tuberculosis, and (in the Centralb. fiir
Bak. und Parasit., August 8, 1895, Nos. 4, 5) Lubinski describes the
utlizatio of this acid broth in the preparation of solid and liquid
mdia, 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 erms. We find that the tuberculosis bacillus will grow upon acid
N
eef broth which requires 21.6 c. c. sodium hydrate solution to neu-
tralize every 100 c. c. of the media. It will also grow upon acid arti-
N
fcial edia every 100 (. c. of which requires 45 c. c. of 10 sodium
ydrate solution for neitralization. This acidity in soime instances
was due to he acids in the meat. In the artificial media it was due to
the acid phosphate of potassiumn 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-
loric acid have beent contained in every 100 c. c. of the media. The
esee of free hydrochloric acid was tested, both before and after the
ei izatio of the media, and after the bacilli had begun to multiply,
by ueas of ongo red or phloroglucin and vanillin. The co io-red
7




8

tes not satisfactory in the beef broth, the al
sibly organic acids present evidently interfering with the reaction.
The phloroglucin and vanillin test, however, proved conclusively the
presence of free hydrochloric acid in the media,u
a good growth of the tuberculoss bacillus The
free hydrochloric acid present in these cultures could not be readily
determined, as when hydrochloric acid was added to the a a po
tion apparently first decomposed the organic salts
After the growth of the tuberculosis germ is wel ad upon
the media described and has apparently ceased, the aid r in
the cultures is still more marked, requiring for the neutrali
of the acid substance produced by the germ to every 10. c. of
N
ture 12.5 e. c. to 15 c. c. 10 sodium hydrate.
Lubinski notes a decided variation in the morphology of the germ as
grown upon the acid vegetable broth. The bacilli, e says, often
appear in long filaments, having, however, the same thickness that t
germs usually possess. The filament forms described by Metshik
Fischel, and Jones varied in thickness, showed side chains, and we
not joined, while Lubinski reports characteristic streptocociike r-
mation. Jones could find this filamentous growth only after the cu
tures were four to five months old. Lubinski observed it in ten days.
OBSERVATIONS.
Our observations upon the character of the germs grown upon id
beef broth media and their morphological changes are somewhat differ
ent. Instead of a thread-like growth or chai growth we have often
found what appeared to be spores, and sometimes the bacilli seem very
much thicker and longer than the ordinary gers.In one culture
examined the germ had been from generation to generation during fur
years grown ion acid media and might be expected to show Lubin~sk's
chain-like formation, but instead there wasa aapparent spoe formation
percepltible.
The appearance of the germs grown upon our acid media can be see
from the accopanying plates. fig. 1, showsthe bacillus grown
upon glycerin beef broth for four months; Neisser's spore stain. P. I,
fig. 2, shows the bacillus grown for forty generations upon an aid
mtedia.
The filament forms are not observed. The is a slight thickening
of thee bacillus and rounding at the end often noted, which would app
to be spore formation. There is a diffeent change in the morphology of
the germ as grown upon our media which confirms the conclusions
Lubinski that we have heteromorphic forms of the tuber lis us
and a genuine pleomorphisius caused by the changed conditio li
Brnms (Centralblatt fir Bak. und Parasitenkunde, Bd. XVII Abt
No. 23) describes a germ which gives Neisser's spore
that particlar evident.sto the ones we ha ve ited







Bul. 13, Bureau of Animal Industry. PLATE I.











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Ia


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'91



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Io,






Fin. 1.-Seventh generation in glycerine heef lroth.
Neisser's spore stain. (> 20:).)












1 I
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L ..e


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/


Fio. 1. Fottieth generaUtion, acidl media.
Nel.isr |Misle stainz L x a:lj.)






Haines, del

BACILLUS TUBERCULOSIS UPON NEUTRAL AND ACID MEDIA.








8I 1 3, Bureau of Animal Industry. PLATE II.

















Sa
|-- ft





I I

0*




FIG. 1.-Culture containing free hydrochloric acid.
Neisser's spore stain. (x 000.)












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e













.i . l 'til hl lllt.r ... Nei.....rs.. l....in.









flttincs, (del.

BACILLUS TUBERCULOSIS UPON ACID MEDIA CONTAINING FREE HYDROCHLORIC ACID, AND
UPON ARTIFICIAL MEDIA.






l. I, fig. 1, shows the germ grown upon acid media containing free
hydrochloric acid.
Sfig. 2, is a drawing of the tuberculosis germ grown on arti-
ficl m We do not here observe quite the same change in the
r gy of the germ.
DETAILS OF EXPERIMENTS.
A moe detailed description of our experiments may be interest-
ig. The reaction of the media prepared for the cultivation of the
t ulosis germ was not changed in the process of sterilization. A
qantitave check of the amount of acid was made before and after

iFirst eerient.-The media used for this experiment was alkaline
d required for neutralization 1 c. 2 hydrochloric acid for every
e0. c. of the media.
a. To eachof 2 flasks 50 c. c. each, 5 c. c. NaOHi were added.
N
b. To each of 2 flasks 50 c. c. each, 20 c.. 10, NaOH were added.
*N
c. To each of 2 flasks 50 c. c. each, 1 c.. ,. 2'Cl were added.
Th six were inoculated at the same time with tuberculosis bacilli
frm (A 14). Within two weeks a good growth of the germ was noted
ovr the surface of flasks a and c, while b showed no growth at all.
S d experimnt.-The media used required for neutralization 8.4
N
c. c. i, sodium hydrate solution for every 50 c. c.
a. To each of 2 flasks 50 c. c. were added 8.4 c. c. -, NaOH.
10
N
b. To each of 2 flasks 50 c. c. were added 1.3 c. c. ,,, 1C1.
: flasks were neutral and b flasks acid. The two sets were iiiocu-
late from the same culture (A 15). Within two weeks the germs in
bot flasks were growing well, a perhaps a trifle better than b. At the
endof seven weeks one flask , was titrated and required for neutral-
i n 6.3 c. c. .0' NaOH. This flask had been neutral when inoculated.
N
ks required 22.5 c. c. NaOl for neutralization. The increase


N
In acd in a jflasks is represented, therrby g.31 e. e. 1,, Na()Iand
Ste b flaks by 7.5 c. c. 10 There was apparently a larger
am1t Of the acid substance tfrmed in the acid flasks than in those
which were netrl at the time of inoculation.
eperi t.-The media used requi fr euralization .6
c NaO I f every 100 c. c. of liquid.
10,8:lR~






10


N
b. To each of 2 flasks 100 c. c,, 2 c. c. !, HCI was added.;v

N
To each of 2 flasks 100 c., 2 c. H. wer
c. To e of 2 flasks 100 c. c., 3 c. c. -2, wOI
These flasks were all ioculated with a virulent ger, n although
it required several weeks for the growth to begin, aftr four w
there was growth in all of the cultures, the most acid ones seeming to
contain as good a growth as the others. very 100 c. c. of the a media
N
had required 11.6 c. c. i, NaOH.

Every 100 c. c. of the b media had required 16.6 c. c. NaO
N
Every 100 c. c. of the c media had required 21. c c. Na fr
neutralization.
Fourth e.peri)ient.-The media use in this experiment was the
artificial media described by one of us-De Schweinitz-in 189, and it
required 45 c. c. 10, NaOH for every 100 c.c. for neutralization.
a 100 c. c. one flask of the media as noted.
b 100 c. c. one flask of the media as noted with the additionof 2 c.
N
2, 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 oth.
Drs. Trudeau and Baldwin have pointed out that the appa ent retda-
tion of the of te th f h tuberculosis bacillus by the acid reaction of the
media can be overcome by eutralization. This is tr; ut we have
also found that if the germ is transferred from an acid culture afer it
has been cultivated on acid media for a number of moths to a etral
nutrient fluid it will no longer grow. There would seem from this to
be probably a poisonous principle secreted by the germ. It may be a
true acid or, more likely, a substanee with an acid reaction. In the
experiment, as the germs used for inoculation still floated o the
surface of the culture they should have been alive une some -
stance especially inimical to their growth was produced i he culture
These experiments show clearly that the tuberculosis bacillus an be
readily accustoimed to an acid nutrient fluid, that it a easly adapt
itself to the changed conditions of life, and tat even a s l a ut
of free hydrochloric acid does not interfere with its g
teud to show further, we think, that under certai conditions there
is probably a poisonous substance produced by the ger w
inimical to their own life.
The adaptability of the bacillus to a variety of m i n est be
explained by spore forl..ation.
MARCH 14, 1896.













FURTHER EXPERIMENTS WITH AN ATTENUATED TUBERCULOSIS
BACILLUS.

By E. A. DE SCHWEINITZ, Ph. D., M. D., and E. C. SCHROEDER, M. D. V.

In the Medical News, December 8, 1894, one of us (E. 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 4 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 4 c. c. of
tuberculosis culture thirty-second generation was repeated, and again
on January 20 1896~, 2 c. 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 live or six days immnediately
preceding death. It had no appetite, the hair was rough, there was
ausea and great thirst and evidence of distress. Post-mnortenm exami-
nation revealed congested lungs and an intlanmmatory condition of
ileum and caecu. Dr. Schroeder says "there were no signs of tuber-
culosis and the small nodules formed at the time of tihe 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 ger.






12

RESULTS OF GUINEA PIG INOCULATION'S

The guinea pig inoculations made by one of us (hreder) with this
attenuated germ to test its virulence were as follows: November 24,
1894, Nos. 280, 21, 22, and 283 received each an abdominal injection
subcutaneously of c. c. tubercle culture twenty-third geeratio.
On January 25, 1895, each pig again received I e. c. tubercle culture
twenty-sixth generation. October 7, 1895, guinea pig N. 281 was folnd
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 intfammation 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 pis,
Nos. 280 and 283, are alive and well at this time.
Guinea pigs HNos. 55, 56 5 and 59 were inoculated on October 2,
1895, subcutaneously with 1 cc. 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 abdomeni. 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 inoc-
lated with .1 c. c. tubercle culture twenty-sixth generation. On Decem-
ber 30, 1895, guinea pig No. 331 was found dead tfom 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 welland show nosymp-
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
tutherculosis.
These inoculations made by Dr. Schroeder have confirmed the earlier
experiments made by Dr. l)e 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 gerr we have succeeded
in obtaining an attenuated germ which, even in large doses, is appar-
ently harmless to guinea pigs, rabbits, cattle, orses, and monkeys.
November 15, 18.%, six guinea pigs were inoculated with c. c. each
of an emulsion of an attenuated culture thirty-niurth genration. The
pigs weighed as obllows:
Ounces. Ounoow.
No. (7 ........................ ...... 9 No. 70 .............................. 1
No. i s ......... .................... 11 No. 71 .................. ............ 8
N ......................... ...... 11 No. 81 .............................. 12








I "ember 18 No. 67 was found dead from pneumonsa. The other
l exhibited the following conditon on subsequent dates:

SDecember 1S, i189.


Guinea pig. Weight. cRematrks.

Ounces.
.................. ...... ............. 12 Slight local swelling; no enlarged glands.
N o. ..................................... 14 Do.
NO.- O ~ --- -- -I ------------------- --------- '13 Do.
. .7 ......-. -.... ........ ...... -...-.. .. .. 13 1 D o.
No.7 -...-............................... 10 Slight local swelling; right inguinal gland cn-
larged.
o.81.....-..--.......-..-.........---......--... 14 Slight local swelling; no enlarged glands.


l)December 31, lS9J.


Guinea pig. Weight. R emark s.

Ovuites.
So. .....-..... ................... ........ 14 Slightly enlarged glands.
N .6--......................... ---- ----- .......--... -16 No enlrgd glandls.
o. 7 .......... ..... ............. ... .. 15 X o.
o. 71...... ............... ..... ....... 11 l)o.
No-71_-----------------------------1 Ito
No. 8 -...........- . ...-.. ............ 14 Slightly enlarged gland.


o tuberculosis was evident on autopsy of one of these, showing
fthatl e germ was without pathogenic effect. The animals had steadily
gained in weight.
Again:


G a pig Weight. Guninea pig. Weight. Guinea pig. Weight.

0nc s. O.I e . .IOu.r .( .
No 1 ................. 15 No. 170................ 14 No. 174 .......... ... 15
No. 1 7. ............... 13 No. 171........ ... ..11 No. 176 ............... 14
o. 18.............. 14 No. 172 -......... ... 16 No. 159 ........ ..... 10
I.1 .....9 ............ 10 No. 173 ............... 16 No. 164......... ....... 10


These anim als were all inoculated on February 5, 18f16, withl 11 c.c.
of attenuated tuberculosis culture thirty-seventh generation. On Feb-
ruary 13, 1896, the weights of these animals were as follows:


Gunea 1ig. Weight. Remarks.

uncttr.
SNo.1S 9 -.--. .........-......-....-...... 9 Swelling size of a Ic, at point of in culation.
172............... ................ .... 15. l.o.
N o .1 ......... .. ......... ... ...... 15 I )o.
N o.172, . .... ... ........... .... . ... 10 l)o.
No.174 ------------------------15 D)o,
No17 ............... .................. Larg swelling at point of inoculation.
N .1 ................................... 14 Slight s elling at point of in ulation.
I N o. 1 s ............. ............. ......... 10 Io.
N o. 173 ............. ..................... 14 i .
o. 1 ... .......................... ...... 8 Slight so ling; pnena nonia.
No. 166. ................................. 1t Do.


On Febrary 8 1d i0, respectively, guinea pigs No. 167 and 176 were
found dead fronm p)neuemn niai, whIich they had contracted alond expso sur
to cold. Subsequently guinea pigs Nos. 1741, 172, 159, and 171 were
rl ;Ir'l







14

found dead from pneumonia. On March 6, 186, gina pig .
which had been inoculated on February 5, with the attenu d
was found dead. There were no signs of tuberculosis, although the
animal had been inoculated over a month. The pig died-from pn -
monia. March 10, 1896, the record of the remaining as was as
follows:

Guinea pig. Weight. Remark

Ounces.
No. 173........................... ............ 15 Slight sore at point of inoculato
No. 169 ........... ....................... 9 No local lesion.
o. 168 ......... ................... ... 16 Slight sore at point of iouatin.
No. 170.. ................ .......... 10 Apparently well.
No.166................................... 14 Do.


Guinea pig. Weight. Guinea pig. Weight. Gui g. Weigt.

March 18, 1896: Ounces. March 23, 1896: Ounces. April 7, 1:
No. 168............ 16 No. 170............... 10 o.168........... 18
No. 170............ 10 No. 168........... 16A No. 166........... 16
No. 173 ............ 14 No.166 ........... 16 No.170 ........... 10
No.166............ 14 No.173............ 13j 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 tuberculosi
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 anials,
its ability to grow in artificial media is as good and better thn 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 TUBER(CULIN INJECTIONS UPON THE MILK OF HEALTHY
AND DISEASED COWS.

By E. A. DE SCHWEINITZ, Ph. 1)., 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 D)r. 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 e. 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 fiiled to show any decrease in the milk
fat. When the rise of temperature was noted in the others a Inarked
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---o. 185-while
the newer cases gave the largest variation. The Tables I and 11 show
the quantity and composition of the various salmples of milk, and the
Tables III and IV 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 siall doses of tuberculin until she ceased to give a reaction and
w 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. c.






Previous to that date she had received altogether 565 c.o
The last ijection caused no cange in the amoun t or
temperature.
The variation in fat should, of course, be attributed n part to
fever. But that this is not the only cause is also evident. The varia-
tion is not, judging from the few tests made, suicient of i to prove
the presence of tuberculosis, but taken in conjunction with the riN of
temperature might be considered as corroborative evidence. The
tuberculin tests were made by Drs. Schroeder and Cur while in the
milk analyses I was assisted by Mr. J. A. Emery.
In this connection are appended several charts with ~~cu s wch
show the average variation in temperatures of a number of diff
animals. These were tabulated in February, 185, fro the reports
received from the different States to which tuberculi been sent.
Chart No. 1 shows the average temperature of a number of anima,
without reference to breeds. The other charts are arranged so tha all
animals of the same breed are placed together. The dia noses were
not in all cases proved by slaughtering the animals, but in all
where they were killed the autopsies confirmed the diagn
The weights of the animals were take s the average weight, and
the dose of tuberculin was 2 c. c. 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 disea
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 tempeiatures
were takei of each animal, and the general average thus obtained with
a large number of animals is given as the average normal temper aure.
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
animals, which were f'rom different herls, miay be due to their larger size.
Many oabjections haveI been made against the use and reliability of
tuberculin as a diagnostic agent, the opposition coming principally
lfom those who are to a great extent unfamiliar with its pratical use
or who are only too ready to condemn a material which, through lack
of skill and knowledge on their part, ha perhaps iven unsati
tory results. The ommiittee in Paris, composed of Cheveau, Leblnc,
Mequin, Nocard, Strauss, Trasbot, and Weber, reported as follows upon
the Irincipal objections to the use of tuberculmn: "The use of h i
tem leratures and carbolic acid in the manufactureof tuberc
it imipossible that the tluberclin, if properly prepared, shold pod
disease. It occasionally happens that tuberculin fails to ive a re
tio in diseased animals but these are very exceptional cases and occur






17

only sometimes when the animals are very badly diseased and their con-
ditiocould 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 fou. 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 c. c. 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 tuberculin 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








18

SI.- nale of ilk of cows inj with tuber

No.of l n -A Albsh Total Volatile
a- Date. Quan cii Total Sug mi- t. in .
mal. tity. g solids. ar. di A t.
real. .... 'oid milk. (a) (b)

1894. e. P.. P. t. P. t. 1. Ct. P. Ct. P. ct. P. t C. c.. :
299 Dec. 11 9,655 1.032 13.65 3.84 3.14 4.48 0.70 .......... 6.7 .13
De. 12 9,655 1.031 14.54 4.17 2.91 6.31 .70 ......... 5.0 .146
Dee. 13 8,519 1.029 13.38 3.84 2.90 5.10 .62 .......... 3.9 .108
Dee. 14 9,087 1.029 13.11 4.16 2.86 4.10 .66 ........ 6.6 .11
Dec. 18 7,950 1.028 12.74 3.33 2.50 5.49 .60 .......... 4.7 .132
145 DIe. 11 2,000 1.018 21.61 5.00 3.21 15.34 .75 .......... 3.5 .100
Dec. 12 1,785 1.025 14.68 2.94 3.13 7.73 .75 .......... 3.9 .136
Dec. 13 3,407 1.026 14.95 2.38 3.49 7.16 .72 .......... 5.0 .130
Dec. 14 3,407 1.027 15.03 3.12 3.51 7.21 .76 .......... 4.8 .13
Dec. 18 3,407 1.027 16.26 2.77 3.75 8.18 .78 .......... 4.8 .114
161 Dec. 11 450 1.020 18.86 2.50 3.98 12.56 1.01 .......... 8 .072
Dec. 12 1,025 1.027 12.99 2.63 3.05 5.52 .77 ......... 5.5 .072
Dec. 13 1,703 1.026 12.77 2.77 2.99 4,09 .74 .......... 7.5 .102
Dec. 14 1,703 1.026 12.92 2.94 3.06 4.91 .73 .......... 6.1 .110
Dec. 18 1,703 1.026 13.73 2.63 3.18 6.35 .81 .......... 5.1 .00
1895.
299 Jan. 3 10,222 1.034 12.61 3.57 2.78 4.26 .73 ......... 3.3 .140
Jan 4 9,087 1.033 12.34 3.57 2.81 3.30 .76 .................... .150
Jan. 8 0,22 1.032 12.45 3.57 2.96 3.87 .69 .......... 7.2 .168
Jan. 9 9.2087 1.034 12.58 3.84 2.13 3.86 .67 .......... 7.2 .1 5
Jan. 10 9,654 1.033 12.95 3.84 .51 4.06 .61 ......... 7.5 .154
145 Jan. 3 3,407 1.028 16.97 2.77 3.36 10.14 .86 .......... 1.4 .100
Jan. 4 3.407 1.030 15.40 3.12 3.05 7.37 .76 .......... 1.9 .156
Jan. 8 3,691 1.030 15.68 3.12 3.56 7,44 .75 ......... 1.9 .122
Jan. 9 3,407 1.031 16.74 2.94 3.86 8.35 .73 .......... 4.9 .138
Jan. 10 3,123 1.028 15.38 3.12 3.58 6.54 .70 .......... 53 .13
161 Jan. 3 1,703 1.030 17.54 2.94 3.48 9.99 .77 .......... 1.4 .130
Jan. 4 1,992 1.027 13.27 3.12 2.97 5.77 .83 .......... 2.0 .132
Jan. 8 2,272 1.029 14.37 2.94 3.20 6.53 .68 ................... 124
Jan. 9 1,988 1.031 14.63 3.33 3.35 6.52 .70 ......... 4.9 .130
Jan. 10 1,987 1.031 14.57 3.33 3.28 5.91 .71 .......... 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 7.7 .132
Feb. 20 8,234 1.032 12.33 3.57 2.63 4.07 .68 3.60 7.0 .150
299 Mar. 4 8,519 1.031 12.29 3.57 2.72 3.87 .67 29.21 7.2 .154
Mar. 5 8,945 1.032 11.99 3.57 2.68 3.46 8 40.63 8.2 .156
Mar. 6 ........ 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 7.5 .156
285 Mar. 19 4,828 1.029 12.84 3.57 3.01 5.14 .67 27.89 5.8 .106
Mar. 20 5,112 1.029 14.60 3.57 3.12 6.73 .67 27.21 5.2 096
Mar. 21 5,396 1.029 13.59 3.57 3.03 5.81 .66 22.9 5.1 .098

a Iodine absorption number.
b Number of cubic centimeters, 1 Ba (oH)2, required for 1 gram of fat.
c Nuiber of cubic centimeters, N Na oH, required for 1 cubc centimetr of milk.
10
TABLE II.-AAnalyses of 7ilk of couw injected with tuberc uln.

185. 189. .22

Iu I e Quan-. SQ Tpecific t Dan- Specifi D speeifi
ity. gravity. Dtte ty. gravity ty. gravity.

e.e. c . c
Jan. 14 5,111 1.029 5.92 Jan. 14 9,655 1.030 4.28 Jan. 14 5,111 024 10.8
Jan. 15 4,260 1.028 5.51 Jan. 15 7.950 1.028 4.29 Jan. 15 5,111 1.24 8.
Jan. 16 4,54 1.030 5. 50 Jan. 1 7, 83 1.030 3. 67 Jan. 16 827 1. 30 5.0
Jan. 17 4,543 1.030 5.71 Jan. 17 7, 67 1.028 4.29 Jan. 17 2 1.02 6.5
Jan. 18 4,260 1.029 5.77 Jan. 18 7,667 1.027 3. 68 Jan. 18 247 1.2 6.12
186. 194. 1119

Jan. 14 J............. ..... .. Jan. 14 1,420 Jan 23 9.0
Jan. 15 375 1.028 5.51 Jan. 15 1,704 1.025 11.06 Jan. 15 5,679 1.022 8.22
Jan. 16 500 1.022 9.04 Jan. 16 1, 98 1.029 6.53 Jan. 16 5,111 127 4.9
Jan. 17 508 1.022 7.19 Jan. 17 2,272 1.030 6.73 Jan. 17 20 1.27 6.9
Jan. 18 440 1.024 6.97 Jan. 18 1,948 1.031 5.09 Jan. 18 16 1 .

187. 195. 24.

Jan. 14 1,08 1.030 8.77 Jan. 14 5,111 1.024 6.01 J 14 5,111 1. 84
Jan. 15 3,123 1.023 12.32 Jan. 15 1,704 1.027 7.97 Jan. 15 4,543 1.23 0.3
.Ja,. 16 3,407 1.028 6.13 Jan. 16 2,272 1.029 4. 4 Jan. 16 1, 30 2
Jan. 17 3,408 1.030 7.51 Jua. 17 1.98 1.028 8.17 Jan 17 3I, 1 7 7
J. 18 3,408 1.031 6.16 Jan. 18 2,556 1.030 6.52 Ja 18 ,111 1.7








19


TABLE I .l-Tetperature reaction. of the cows used in the milk experiments.


Date. Time. No. 299. No. 161. No. 145.


1894.

Before injection. o o
December 11 -- --.................................................... 1.00 p. m. 102.3 101.3 101.6
3.00 p.m. 102.0 98.2 97. 3
5.00p. m. 101.5 99.4 98.5
After injection.
ber2 (ijet 2 c. 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
2.00 p. in. 101.0 102.0 101.2
4.00 p.m. 101.4 101.0 101.6


Before injection.
January 7 .......... ....................................... 1.00 p.m n. 101.0 102.4 100.6
3.00 p.m. 101. 0 102.6 101.2
| 5.00p.m. 101.2 102.7 101.4
A 'fer injection.

January 8 (inected 2 c. c. tuberculin at 8 p. n. January 7 ....... 8.00 a.nm. 101.5 101.8 105.2
10.00 a.n. 101.0 101.7 105.0
12.00 m. 100.4 100.8 102. 6
2.00p.m. 101.1014 01. 0 101.5
4.00 p. m. 101.3 101.5 101.1
Before injection.
Fbrary18....... .... ......................................... 9.00a.m. 101.6 ........ .......
12.00 n. 100.6 ........ ........
4.00 p.m. 101.9 ................

After injection.

February 19 (injected 2 c. c. tuberculin at 9 p. im. February 18).. 8.00 a. im. 101.6 ................
9.00 a. m. 102.7 ...............
10.00 a. m. 102.6 ........ .......
11.00 a. m. 100.2 ....... .......
12.00 100. 2 ...............
1.00 p. m. 101.2 ...............
2.00p.mn. 101.5 ................
3.00p.n. 101.8 ....... .......
4.00 p.m. 101.8 ..............
5.00p.m 101.4 ...............
iBefore injection.
March .................................. ................... 8.30 a.m. 101.2 ..............
12.00 m. 100.2 ................
r j 4.30 p. n. 101.2 ................
After iitection.

Mrh (injected 30 c. tuberculin March 4).................... 8.00 a.m. 101.5 ..
9.00 a. 102.0 ........ ........
10.00 a. 99. 2 ...............
12.00 1002 .......
2.,00p.m. 101.2 ................
4.00p.i. I .5......
5.00p.m. 101. ..............
.. .... . ....... .... . ........... .. ---- ...








20



[First test of tuberculous cows.]

No. No. No. No. No. No. No. No. No.
Tm
185. 186. 187. 189. 194. 1929. 231.

1894.
Before i .ojectitin.
0 0 o O 0 0 0 0
ember7............. 9.00p.i. 102.0 100.5 100.2 101.5 101.2 102.5 102.2 101.3 1.9
After injectio.
December 8 (ijected 21
c. c. tuberclin 11 p. i.
ecember7) .......... 8.p.mi. 102.4 101.2 101.3 102.0 102.0 105.8 102. 105.4 103.
9.0p.m. 3 103.1 101.4 100.3 101.8 103.8 107.0 103.6 104.8 103.6
10.00p.m. 105.3 103.2 102.4 104.8 104.0 107.2 104.6 103.4 103.8
11.00p.mn. 105.6 105.6 102.1 105.8 1038 107.2 106.4 10.2 104. 8
12.00m. 106.6 106.0 101.5 106.6 104.3 107.4 10.2 106.9 104.
2.00 a.im. 106.0 105.6 99.7 106.4 106.1 106.7 106.5 106.5 105.2
4.00a.m. 105.4 105.1 104.7 105.6 10.2 105.3 105.0 105.4 105.7
8.00 an. 103.0 106.2 104.8 105.6 1020 104.2 105.2 104.4 104.7
10.0 .a.m. 101.0 100.5 101.0 100.5 101.0 101.1 101.0 1004 02.


[Second test of tuberculous cows.]

1895.

fre injecti.1022 102
January15............ 4.00p. nm. 102.2 102.5 101.8 102.8 102.0 101.4 101.4 101.6 100.2
9,00p.m. 102.0 101.8 100. 8 101.8 101.0 101.0 101.4
After injection.
January 16 (injected 2
c. c. tuberc lin 11 p. m.
January 15)........... 8.00 a. in. 101.5 102.0 99.6 10 102 .6 105.2 104.7 103.2 105.3
8.30 a.m. 101.8 103.2 101.0 1020 101.8 105.2 105.2 104.0 10.4
9.00a.im. 102.4 103. 2 101.2 103.3 102.6 105.2 105.2 104.6 105.4
9.30a.m. 102.2 103.5 101.2 103.2 102.8 105.3 104 10.4 105.8
10.00a.ni. 102.2 103.6 101.2 103.4 102.8 105.4 10.3 10 106.0
10.30a.m. 102.2 103.0 101.6 104.2 103.0 105.2 106.0 105.5 106.
1.00a.in. 102.4 103.2 101.5 104.6 104.2 105.1 106.0 105. 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 10.6 106.3
12.30p.m. 103.6 102.6 103.6 10 004. 105.5 104.8 106.2
1.30p. m. 103.5 103.0 103.9 104.8 104. 1045 10.5 1.2 106.0
2.00p.m. 103.4 102.0 104.5 104.8 104.6 104.0 104.6 104. 10.6
2.30p.m. 103.6 101.6 104.6 104.4 104.2 104.0 103.4 103.5 10.2
3.00 p. 102.4 100.0 105.0 100.0 102.8 103.4 1.5 1024 104.
3.30p.im. 101.9 .5 105.0 99.6 102.4 103.0 103.2 102. 104.2
4.00p.m. 101,6 99.3 104.6 99.4 102.0 103.2 103.2 102.6 14.
4.30p.m. 102.8 99.6 104.5 99.4 13 1 104.2










FiG. 1.-Chart showing average temnperature of 3,910 healthy and 1,l.' diseased cattle inCjected with tuberculin. (a)
(Average normal temperature of 1,191 tuberculous cattle before injection, 101.6o F. Average normal temperature of 3,930 h vealtlhy attle before iujection, 101.5 F,1

FRA//R. HOVAS AFTZR /IJECT/ON
/0' 6 8 /0 i2 14 16 /8 20 22 24 6 28

.6















.8 h


.6







a Tabulated il February, 1895. b Temperature at time of inje tion.
ii. _. .._ .. ... .. . .
.4irl __ _ _ _ _ _ _ _ _ _ _ _ _




.2"";"" ____ ___ __ _ _ _ _ __ _ _ _ _ _ __ _ _ _ _
/010" __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
.8 __ __ __ __ __ __ __ __ __ __
.6iiii;,, n~o,,
.11: 11',,i,; il, "il" ii. ;i i;; i;
0;;;,, ;; ,,;,,,; ,;;, ,'i;i








fG. 2.-Cart showing areage temperature reaction of 62 tuberculous and 93 healthy registered Jerseys; 71 tuberculous and 303 healthy grade Jerseys;
9S tuberculous and 195 healthy Jerseys inijected with tuberculin. (a)
Average normal tperature of 231 tuberculous cows before injection, 101.4 F. Average normal temperature of 591 healthy cows before injection, 101.7o F.j

fA HOURS AFTER. INJECTON.
6 8 /0 12 14 /6 /8 20 22 24 26 28

107"


106'
.5
105 "__ ___ ______-




*5W ___-__ _______ ______________ _______
.5 0.0
0





II




a in ..... b T..peratur at ti.e. of ij .c.. ..








FIG. 3.-Ch art 8towing arerage temperaturc reaction of 40 tuberculous and 68 healthy grade iolsteins; 70 lubercutlot0s and 104 healthy Histeins injecre
with tuberculin. (a)
[Average normal temperature of 110 tuberculous cows before injection, 101.8 F. Average normal temperature of 172 healthy eows befoe injection, 101.8 F.

HOURS AFTER INJECTI/ON
FA 6 8 /0 12 14 16 18 20 22 24 26 28
107,
.5
/06
- ~ ~ ~ 0 _ _ _------------__ _ __\-----------





.5

w u ----y----\--
104"
/4 _____________/ ^________ ________\- ___ ____

S --_yr----------
103"






.5

a Tabulated in February, 15. b Temperature at time of injection.


,1 .I .. .: .......








FiG. 4.-Chart showing arerage temperaturhams;e reactioSn f i tubrc o an 64 hea uras; 5 tuercu and 11 healthy gradae Dirhams injected
with tuberculin. (a)
Average nor tenmlature of 32 tuberculous cows before injection, 102.10 F. Averge normal temperature of 75 healthy cows befoe injection, 1I01. F.]

fAI 0HOURS AFT7E IN/ECT/ON.
f 6 8 /0 12 /14 /6 /8 20 22 24 26 28

107'

.5

0 ______ _______ _______ _______ _______ _______ _______




.5 _
04
.5 ___


103 c'
.5

1020'




S..5

a Tabulated i February, 1895. b Temperature at te of u jection.


........ ..... .,









FIG. B5.-Chart showing average temperature reaction of 15 tuberculous and J2 healthy .1yrshires injected with Inbercuin. (a)
[Average normal temperature of 15 tuberculous cows before injection, 101.6 F. Average normal temperatue of 24 l(althy 1ows b IFur ijection, 10t.2 F.

FAHR. HOURS AFTER IN/ECTION.
6 8 /0 /2 /4 16 18 20 22 24 26 Z8


.5
1060
.5
105


04 _______ _______


.5
/ .5 1 1
/02

.5 _____**__*"'" _*______* __ _ ___ __ ___


.5
S/ u0
a. Tabulated i Febriuary, 1895. b emperaiture at timue of inje ction.










FIG. 6.-Chart showing arnrage temperature reaction of 6; tuberculous and 108 Itealthy Guernmeys injected with tuberculin. (a)
(Averge normal tiemperauture of 6 turculous cows before injection, 101.10 F. Average noral temperature of 108 healthy cows before injection, 101.70 F.

... 6HOURS AFTEZI /NJECTION
H 4R. 6 8 /0 /12 /4 /6 /8 20 22 24 26 28

107"

.5 '_____________ _______ ________________________ _______
.S



/05





.s
0
/-t .....------. ......----

5_



10 I __________ ____________________ __________



.5 b e--- - -- -

10/
.5



a Tabulated in February, 1895. b Temperature at time of injection.


.. .
mmg~g. - ....... ....... ... .








FIG. 7.-Chart showing arerage temperature reaction of 241 tuberculois and 537 healthy grades injected with tuberulin. (a)
[Average normal temperature of 241 tuberculous cows before injection 101.8 F. Average normal temperature of 527 healthy coiws befor injection, 101.5 F.]

SHO-URS AFTER INJECTION.
IAR. 6 8 /0 /2 14 /6 /8 20 22 24 26 28

1070




.5 _______ --_____________________
/05'" S"
1040







1020






.o o .... . . . .. ..... ........ .. .
& ---- --- .. .........**


5... .. . .. ... .. ..... .. ... . .. . ... .. . . .



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/0/;,,, _______ __ __ ________,; _; 51;





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