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Temperatnre at 11^ a. m. (one hour aft^, 100.6° F.
Temperature at 1.30 p. m.^ IHO.T F.
Temperature at J^ p. m., 102.4** F.
Temperature at 4 p, m., 102.2° F.
During this above period breathing was rapid, with an occasional rigor.
Guinea pig No. 422 (weight, 284 grams) received 0.0095 gram of crystals at
12.05 p. m.

Temperaimze sA time of injection, 99.8° F.
Temperature at 2.30 p. m.,97.4° F.
On the next day there was quite perceptible swelling where the injection was
made. Pig was chloroformed at end of twenty-four hours and showed consider-
able inflammation at seat of injection. Tissues were hemorrhagic and bathed in
a serous exudate. The muscular tissue was much disintegrated, resembling the
appearance from the action of a caus&.

^ Guinea pig No. 511 (weight, 183 grams) received 0.0048 gram in 0.5 cc. water
at 11.25 a.m., subcutaneously in thi^

Temperature at time ot injeci^n, 103° F.
Temperature at 12.25 p. m. (one boor after) , 101.8^ F.
Temperature at 1.15 p. m., 102° F.
Temperature at 3.25 p. m. , 100.8° F.
Chloroformed next day. Considerable inflammation, with serous •zndate at
seat of injection.

^ New York Med. Journal, March 11, 1882.

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Gmnea pig No. 10 received 0.0274 gram at 10.10 a. m.
Temperature at time of injectiiHi, 90.2° F.
Temperature at 10.40 a. m., 100.2° F.
Temperature at 11.15 a.m., 100.6° F,
Temperature at 11.50 a. m., 100.6° F.
Temperature at 3 p.m., 100.2° F.
During above i)eriod this pig showed signs of restlessness, breathed rapidly, and

//. Tuberculous giUnea pigs.

Guinea pig No. 181 received 0.017 gram at 10.25 a. m.

Temperature at 11.40 a. m., 101.4° F.

Temperature at 1.50 p. m., 102.8° F.

Temperature at 2.50 p. m. , 108.4° F.

Temi)erature at 3.50 p. m., 103° F.
Pig sat drawn up in cage and shivered.

Guinea pig No. 259 had received virulent tuberculosis two weeks previous to
injection of crystals. Received 0.0172 gram at 10.45 a.m.

Temi)erature at time of injection, 102.4° F.

Temperature at 11.45 a.m., 101.6° F.

Temperature at 3.20 p. m., 101.6° F.
Distinct rigors and rapid breathing.

Quinea pig No. 377 inoculated wit^ sputum from tuberculous patient some
time before injection of crystals. Received 0.023 gram at 11.35 a. m.

Temperature at time of injection, 101.2° F.

Temperature at 12.35, 100.6° F.
Trembling very noticeable.

Guinea pig No. 11 had been inoculated with attenuated and virulent tubercu-
losis culture (weight 448 grams). Received 0.0096 gram of crystals at 11.25 a. m.

Temperature at time of injection, 103° F.

Temperature at 12.25 p. m., 100.8° F.

Temperature at 1.15 p. ul, 101° F.

Temperature at 3.25 p. m., 100.8° F.

The idea was suggested from these experiments that this acid, evi-
dently a secretion of the bacillus, was one of its most powerful weap-
ons; that by its action upon the tissue the cells were first destroyed,
so that they could subsequently be utilized by the germ as food, and
in this way the germ protected itself from surrounding leucocytes.
To test this, crystals dissolved in sterile water were injected by means
of a hypodermic syringe directly into the liver. At the same time an
equal quantity of water was injected into a check in the same way.
After forty-eight hours check and experimental animals were killed.
The check failed to show any effect, while the other exhibited a liver
with several light spots. A repetition .of this experiment gave the
same results.

No effort was made to recover these crystals from the liver, as the
amount used was too small. We did not test the effect upon the liver
by an intravenous injection, as would otherwise have been done,
iK^cause we had found that there was a combination of this acid sub-
stance with the albuminoids or bases, and any intravenous injection
would have resulted in its immediate conversion into a modification

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by uniting with the albuminoids in the blood. Further, the growth
of the germ in the body is localized, and where localized the necrotic
areas are apparent, so that the fairest test was to bring the substance
as soon as possible in contact with the tissue. The experiments in
injections of the animals and appearance of sections follow:

Injection of crystals from artificial cultures of BaeiUus tuberculoids into the liver

of guinea pigs.

Guinea pig No. 409 received X).00178 gram in liver on left side at 11.45 a. m.
Pig weighed 838 grams.

Temperature at time of injection, 101.6** F.
Temperature at 8 p. m., 102** F.

Chloroformed October 24, 1896, at 12 m. Liver dark, with one or two small
white spots in it, and apparently a small inflamed spot at about the position
where injection was made. Gall bladder injected and seemingly inflamed.

Guinea pig No. 412 received 0.0037 gram in liver at 1.45 p. m. Pig weighed 242

Temperature at 1.45 p. m., 103'' F.
Temperature at 3.80 p. m., 100.4"* F.

Chloroformed at end of forty-eight hours. Gall bladder was congested (not so
much as in No. 409). Liver showed pale si)ots and one or two small white areas
of apparent necrosis. Hardened in HgCl, on microscopic examination one area
rather well defined where the liver ceUs do not take hematoxylon well, though
stained nuclei could be seen.

Checks on injection of crystals into liver.

Guinea pig No. 510 received 0.25 cc. sterile distilled water in liver.

Chloroformed after forty-eight hours. Post-mortem: All organs, liver, lungs,
spleen, etc., normal.

Guinea pig No. 387 received 0.50 cc. sterile distilled water in liver. Chloro-
formed after forty-eight hours. Post-mortem: All organs normal excepting one
or two very small pale spots in liver ; no necrosis.

Injection of crystals. Guinea pig ** C " received 0.0043 gram in liver. Chloro-
formed after six days. Pig weighed 600 grams.

Lungs very slightly congested. In large left lobe of liver there were two or
three comparatively large areas of necrosis. These sx)ots were on the side in
which injection was made, and the Hver appeared to show the track of needle.
The guinea pig was otherwise healthy.

Guinea pig *' E " received 0.0023 gram of crystals in liver. Chloroformed after
two days. Pig weighed 345 grams.

All organs appeared normal, except stomach and liver. The stomach showed
a slight inflammation in its wall on the side which lay next to a necrosed spot in
the liver.

Besides this sjmt there were several others of considerable size in the liver on
the side on which the injection was made. The section of pig **C," the one
allowed to live six days after injection, showed on microscopic examination tue
following :

Stained with hsematoxylon and eosin ; distinct areas of necrosis were noted, the
most marked ones near the surface of the liver. Polynuclear leucocytes were
present, though not in large numbers, in and around the necrotic areas, and there
was also an increase in connnective tissue cells of the liver around these same
areas. Plate VII is a drawing of the liver section, showing healthy and necrosed

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Prudden,^ 1892, suggests that caseation, so constantly present in
tuberculosis, is probably due to a specific metabolic product of the

It seems very reasonable to conclude from our experiments that we
have here the substance formed by the bacillus which is responsible
for this necrosis.

The formula which can be deduced from the analysis makes this
acid correspond closely to teraconic, which has properties very simi-
lar to those noted by us in connection with this new acid. Its identity
we have not yet proved or disproved, as the preparation of teraconic
acid is not completed. The amount of this acid obtained is very
small, so that we have used only a very small portion of it for testing
its immunizing property. A single injection of 0.0020 gram was suf-
ficient to keep the animals alive some weeks longer than the checks,
and its solution appeared to exert some slight bactericidal influence.

As this substance seemed to be a temperature-reducing principle
in healthy and diseased animals, we endeavored to separate the fever-
producing principle independently. The crystals were always found
in the culture liquid, and only minute amounts could be obtained from
the bacilli themselves that had been grown on liquid media. Accord-
ingly, these bacilli, carefully filtered without heat, wei*e washed in
cold water, and next extracted with hot water. This hot water
extract contained an albuminoid which caused the tuberculin reaction
in tuberculous guinea pigs and calves upon repeated injections.

Roux and Nocard^ state that they have a tuberculin which will
give reactions almost indefinitely, but do not describe its method of
prepai-ation. Whether this is the same substance that we have
obtained we are unable to say, but certain it is that the tuberculin
prepared in the way we have indicated will give reactions four or five
times in succession, where the reaction with tuberculin as prepared
in the ordinary way fails after the second time. The conclusion is a
fair one. We think that the fever-reducing principle having been
removed, to an extent, if not entirely, the immunity from the fever-
producing principle is much more slowly acquired. Our tests upon
guinea pigs and tul^erculous calves were made with only one day
intervening between the injections. (See Table 1.)

In the Deutsche medicinische Wochenschrift for April 1, 1897, Dr.
R. Koch^ describes some new tuberculin preparations. The dried
tuberculosis bacilli were taken (the culture medium used is not men-
tioned), finely powdered and centrifugalized with distilled water.
The opalescent solution obtained, tested upon animals, gave the
tuberculin reaction. The residual germs were submitted to this treat-
ment a number of times, until finally all were practically dissolved.

' New York Med. Journal, September 10, 1893.
' « Receuil de m6d., 1897.
»Dent. med. Woch., April 1, 1897.

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The letter solutions in lai-ge doses eansed a renetion, but in small
quitiitities) did not prodoee tbts result, and seemed to exert both an
immunizing and curative action in-experimental tuberculosis. Koeli
used for this work virulent germs, and elaims tkat attenuated germs,
do not give au active product. My own work was done with germs
purposely attenuated by cultivation, and the results show that very
active fever-producing, ferer-reduciBg, aad probably curative princi-
ples can be obtained from them. It hardly seemed justifiable to
myself or others to powder dried virulent germs and have the dna^
floating in tlie air. Koeh further refers to two fatty aeids which, in
conjunction with Proskaner,^ had be«n found in the bodies of thie
germs. The writer * of this paper published in the American Chemi-
cal Journal for August, 18^5, a preliminary sindy of the fats of the
tuberculosis bacilli, showing the high content of fat in the bodies of
these germs, which accounts for the diflfteulty in staining them witfe
certain colors, as well as their difficult absorption.

In a later paper, 1806,^ we described briery the different acids
obtained from the body of the germ, both high-melting and low-melt-
ing acids,^ Ixrt whether or not these are identical with those observed
by Koeh and Proskauer we can not tell from the brief mention made
of them.

From otir result it seems very reasonable to think that the necitjtie^
acid is the fever-reducing principle, the albuminoid the fever-pro-
ducing principle, and the reason the tuberculin ordinarily does not
react continuously is on accoopt of their presence at the same time.
At any rate, tuberculous guinea pigs tested successively with tuber-
culin .showed no reaction, while with this albuminoid, which we will
can cell extract, a reaction was obtained.

The i)reliminary exi)eriments published by the writer in 1894* npom
the production of an immunity or resistance to tuberculosis by attenu-
ated cultures have been continued and are eonllrmatory of the first
results, showing the production of great resistance and in some cases
complete immunity. A detail of two sets of these experiments may
be given as an instance of their general results (Table 2).

The first effect of the injection of the attenuated germ was in some-
instances to cause a slight decrease in weight; sometimes a local
swelling was noted at the point of injection, and occasionally an
enlargement of the inguinal glands. This disapi>eared after some
weeks. This local swelling is probably due to the mechanical action
of the bodies of the germs, on account of their high fat eontent and
possible presence of a minute amount of the acid, earning necrosis.

' Deut. med. Woch., April 1, 1897.

'Journal of the Amer. Chem. Society, August, 1895; Bulletin No. 13, 1896,
Bureau of Animal Industry.
» Centralb. f. Bak. u. Parasit., vol. XIX, 18, 19, 1896.
4 Med. News, December 8, 1894.

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It does not skLwmyn result from a subcntaneoos mocnlation, and an
ftpf^arent immmuty from this action is acquired by repeated injections.
Tlus is well siiown in. horses and eows submitted to treatment with
the attenuated ^erm.

From six to eight weeks after the date of the injection of the
germ gumea pigs seem to be entirely well, and are then inoculated
with the virulent germ. As can be seen from the chart, the cheeks
died within six weeks from date of inoculation, while the ones vacci-
nated remained weB loor motnths afterward. It has appeared from
■aany expenuents that if the inoculation with the virulent bacillus is
Biade before complete recovery from the treatment with the attenuated
bJMilhts the resistanee is considerably less. The inoculation of the
animals with the virulent baeiBus,^ and subsequently with a single
tejeetioii of the attenuated bacillus, i^unred that the latter produced
a slight resistance, but ik> very material retardation of tiie disease.

The production of this partial immundty Otr artificial resistance by
means of the attenuated cultures suggested already, in 1894^ the
availability of t^ds same materia]; for tbe pnrpose of treating animals
for the production of a serum which would have some effect in
eurii^ tuberculosis. It suggested the idea further that possibly
cattle could be vaccinated wrih this attenuated germ and made
immune from tuberculosis*

Two cows and (me heifer were selected for the work, which was
eonducted for us by Dr. Schroeder, in charge of the Experiment
Staticm of the Bureau of Animal Industry. One of these animals
was oxiginally tuberculous^ the other two healthy. The tuberculous
a^T"*^^ received large doses of tuberculin until it had received alto*
gether 19,407 ce. (19^ liters), and as much as 1,500 cc. of tuberculin
at a single dose, from November, 1894, to April 20, 1897. The other
animals received injections of the attenuated culture, the amount
injected in fifteen mooths being 11,425 cc. and 18,100 cc., respec-
tively; and by this is meant the liquid culture media in toto, includ-
ing the germs, just as taken from the incubator, without any further
treatment. At first the injections produced a slight reaction and
occasKNiaUy a local edema and abscess. After they had been con*-
tinued for some time this effect diminished or disappeared. The
lerum of aB of these animals was tested a number of times. Guinea
'pigs were injected with the serum in quantities varying from 1^ to
6 cc, and subsequently inoculated, together with the checks, with a
culture sufficiently virulent to kill the checks within four or five
weeks, or the pigs were inoculated with the virulent bacilli and
treated by subsequent injections of the serum. Without giving the
detail <rf the experiments, we may say that the serum from the cow
treated with tuberculin would cause in the pigs a slight resistance t^
the disease; the serum ei those treated with the attenuated bacilli
produced more resistance on the part of the animals or prolonged

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their life to some extent, but not sufficiently, as compared with the
quantity of material injected, to make the use of cow serum appear
practicable. The cow serum, although sterile, frequently produced
abscesses in guinea pigs. This serum we exi)ect to test again shortly,
when it should be more potent.

While these experiments were in progress two horses had been
pressed into service. They were treated by injecting the attenuated
cultures, culture fluid, and bacilli. The first injection of 5 cc. caused
a decided temperature reaction, local edema, stiffness, slight loss of
appetite, recovering after a few days. At first local abscesses were
formed, which healed fairly readily. After a time the abscess form-
ation ceased or occurred occasionally. After eight months' treatment,
the doses of the culture being gradually increased up to 300 to 400
cc. — the total amount injected in fifteen months = 4,459 cc. — the
serum was used for testing. It separated out clear and well. Two
sets of illustrations may be given to show its action on tuberculous
animals. (Table 3.) In one set the checks and two treated pigs died;
the other two treated pigs are alive and in perfect health, apparently,
after a number of months. In another set the checks, four in num-
ber, died within four to five weeks, while the treated ones lived two
or three weeks longer, showing, on autopsy, much less disease in the
lungs than the checks. We endeavored further to isolate from the
serum antitoxic substances by a slight modification of the Brieger-
Boer method. We finally succeeded in obtaining a small quantity of
a grayish powder giving the biuret reaction, soluble in water with diffi-
culty, which was used for treating guinea pigs in the same way as the
serum. The result was as in the first instance. The pigs, one-half
pound in weight, were inoculated with a virulent germ and treated by
a single injection of 0.008 gram of this solid substance. The}' lived
three or four weeks longer than the checks, again showing considerably
less disease in the lungs and less necrosis was noted in the liver.

The effect of the serum was also tried in preventing the rise of
temi)erature in tuberculous guinea pigs, and in saving them from a
fatal dose of tuberculin. As can be seen from the temperature
reactions in Table 1, the injections of one-fourth cubic centimeter of
diluted tuberculin, and at the same time of one-half cubic centimeter
of the serum, either caused a decided reduction of the temperatui*e or
prevented a characteristic tuberculin reaction in animals weighing
about 400 grams. This is one way of gauging the serum.

The result of aU this work leads us to the conclusion that the
injection of the live culture produces substances antitoxic to tuber-
culosis; that the quantity of this substance can be increased gradually;
that the treatment of tuberculosis is and will be for some time still in
the experimental stage. One point, however, must be remembered,
namely, that while it may be difficult to cure the disease in a guinea
pig, where its course is very rapid — a virulent bacillus requiring only

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from four to five weeks to kill — it might be much easier to cheek the
disease when more prolonged in action, as in the majority of cases in
man. Again, in addition to some form of si)ecific treatment for the
disease, man usually has the advantage of being placed under the
best possible surroundings as to diet, climate, etc., and every effort
is made to aid the improvement of the patient, while with experi-
mental animals the conditions are different.

The experimental results obtained lead undoubtedly to the con-
clusion that while the treatment with antitoxic serum is still in the
exx)erimental stage, and should as yet be used only in sanitariums
and under the best conditions, we are on the road to success in the
treatment of this disease and nearer our goal than ever before.

In an experimental way, the antitoxic serum as prepared in my
laboratory has been used by Dr. Stubbert at the Loomis Sanitarium,
and some by Dr. Trudeau at Saranac Lake, as well as by Dr. Charles
W. Richardson in Washington.

The serum used for these cases was of the same lot that cured the
guinea pigs reported in Table 3. The treatment has not been con-
tinued long enough for positive conclusions to be drawn. One of the
cases reported by Dr. Stubbert has been apparently cured, and the
others much improved. Dr. Charles W. Richardson reports decided
improvement in the cases upon which this serum has been used. Dr.
Trudeau has not used the material long enough to come to any defi-
nite conclusions. He noted reduction of high temperatures under its
influence in one of the cases tried.

Maragliano, Babes, and Behring are the other principal workers in
the preparation of an antitoxic serum for tuberculosis. Paquin also
has an antitoxic serum on the market.

Maragliano ^ gives the method he has used for the production of
antitoxic serum, and notes that there is present in the cold-filtered
cultures of the tuberculosis bacilli a substance which causes the
reduction of temperature, and another, not destroyed by heat, which
causes the rise of temperature. In all probability, without isolating
the principle, Maragliano was using solutions of the crystalline sub-
stance we have described in the beginning of this paper. While this is
not destroyed by heat, as he seems to think, it does undergo some change
by combining probably with the albuminoid matter in the media, and
thus losing its distinct property as a temperature-reducing substance;
or, more probably, its temperature-reducing property is disguised by
the presence of the temperature-producing principle extracted by hot
water. The serum which he obtains from treatment of the animals
with the different products of the bacilli is claimed to have some effect
in reducing the temperature and apparently improving the disease.

In the Zeitschrift ftlr Hygiene, Babes,^ reviewing a portion of the

» Revue de la tnberculose, juiUet, 1806, p. 131.
'^Zeit. f. Hygiene, Bd. xxiii, Hft. 3.

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work upon the treatment of tuberculosis with serum, comes to the
conclusion tliat he is the first individual to have discovered any anti-
toxic properties in this serum; that there is an antitoxic substance
present in treated animals, but that it has not yet been brought to a
sufficient development to warrant general use.

Our experiments lead us to conclude that while the injection with
tuberculin in healthy animals produces a serum containing antitoxic
material, the amount of this is small, and that the injection of the
live culture is the proper treatment. We can not agree with the state-
ment made that horses are unsuitable for the work. Mules and
donkeys may perhaps give quicker results, but horses seem to be emi-
nently satisfactory. At no time have we found that the horse serum
produces toxic effects, although this has been noted in the cow serum.

If the antitoxic serum treatment for tuberculosis could be freed for
the present from its commercial aspect, and careful, systematic experi-
ments continuously conducted in numerous hospitals and sanitariums,
this or a similar modified method of treatment could be looked to for
good results. When tuberculosis can be uniformly cured in guinea
pigs as certainly as diphtheria, then does the commercial aspect become
a fair and legitimate one.

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Table 1. — Test of cell extract, tuberculin, and aerum.


No. of



Substance injected.



a. m.

pi m.

p. m.


















i cc. tuberculin + k cc. serum.
i CC. tubeppuHn










Attenuated, tu-


guinea pig.
do —

Attenuated, tu-

i ce. taberculin + i cc. serum.

2cccellextraet = OLOOiOgram.
1 cc. csll extract = aOttSO gram .

i cc. tuberculin








; loao





. 103.8


', 103.8


Online LibraryUnited States. Bureau of Animal IndustryAnnual report of the Bureau of Animal Industry for the year ... → online text (page 34 of 69)