THE ATTENUATED BACILLUS TUBERCULOSIS 
ITS USE IN PRODUCING IMMUNITY TO 
TUBERCULOSIS IN GUINEA-PIGS. 
BY 
E. A. De SCHWEINITZ, Ph.D., 
BIOCHEMIC LABORATORY, BUREAU OF ANIMAL INDUSTRY, DEPARTMENT 
OF AGRICULTURE, WASHINGTON, D. C. 
FROM 
THE MEDICAL NEWS, 
December 8, 1894. [Reprinted from The Medical News, December 8,1894.] 
THE ATTENUATED BACILLUS TUBERCULOSIS: 
ITS USE IN PRODUCING IMMUNITY TO 
TUBERCULOSIS IN GUINEA-PIGS. 
By E. A. DeSCHWEINITZ, Ph.D., 
BIOCHBMIC LABORATORY, BUREAU OF ANIMAL INDUSTRY, DEPARTMENT 
OF AGRICULTURE, WASHINGTON, D.C. 
APPARENT IMMUNITY TO TUBERCULOSIS IN 
GUINEA-PIGS. 
It is well known that a number of the pathogenic 
bacteria decrease very greatly in virulence when 
cultivated for a long time outside of the animal 
body. This fact has also been noticed in the case 
of the bacillus tuberculosis, and the literature is 
full of references to the decrease in virulence of 
the tubercle bacillus and the way of securing this, 
but some facts have been developed in this connec- 
tion in my work on tuberculin, in a new direction, 
which should be recorded. 
Cultures with which I started several years ago 
were obtained through the kindness of Dr. Trudeau, 
of Saranac Lake, N. Y., and were the second 
generation on blood-serum from a rabbit. After 
growing for several generations upon glycerin-agar, 
about two months to each generation, the culture 
was transferred to glycerin beef-broth, which was to 
be used as a source for tuberculin. After the four- 
teenth generation I noticed that the guinea-pigs 2 
that were always kept inoculated with tuberculosis, 
to test the tuberculin, did not succumb nearly as 
readily, and in the case of the fourteenth generation 
it required six months before the disease developed. 
I, consequently, inoculated a number of guinea-pigs 
with the seventeenth, eighteenth, nineteenth, and 
twentieth generations of this germ, which was alive 
and would grow very readily on the liquid media. 
Some of these pigs inoculated had previously been 
treated by an injection of tuberculin; the others 
had never been used. After some months those 
that had been inoculated with the attenuated germ 
remained quite well, and one was chloroformed, and 
the autopsy showed that it was free from disease. 
In order to see if this might be accidental I then 
had nine pigs inoculated, four checks, four pigs 
previously inoculated with the attenuated germ, and 
one pig that had received tuberculin by feeding. 
These were all inoculated in the side with the material 
from the gland of a tuberculous cow that had just 
been killed. After seven weeks the checks were all 
found dead from tuberculosis. The other animals 
appeared perfectly well. One was accordingly 
chloroformed, and although very careful examina- 
tions were made no disease could be detected. Even 
the local lesion that was produced where the material 
had been injected had entirely healed. The animals 
were apparently immune. 
Some experiments that had been made earlier 
with tuberculin prepared from a younger growth 
did not show that this material had a positive value 
in preventing disease, although it retarded it. As 
the preparation of tuberculin, however, continued, 3 
I noticed that the tuberculin was much more con- 
stant in reaction and that a larger amount of the 
active principle was yielded by cultures of the older 
germ. 
At this time I had at the Experiment-station ot 
the Bureau the calf of a tuberculous cow, which, 
however, although the mother was badly diseased, 
did not respond to an injection with the tuberculin. 
The calf was then drenched with ioo c.c. of a 
growing active liquid culture of this attenuated 
germ, the twentieth generation. After three months 
the calf was again injected with tuberculin, and, fail- 
ing to respond, was killed. The autopsy showed the 
animal to be perfectly healthy. 
Another healthy Southern cow received into a 
vein an injection of the same generation from this 
liquid culture, in July. On November 20th this 
animal, which was rather thin and had an initial 
temperature of 103° F., was injected with tuberculin, 
but did not show any reaction. Another animal 
known to be tuberculous, injected at the same time, 
with the same-sized dose of tuberculin, showed a 
good reaction. The cow which had received the 
injection of attenuated culture had a small lump, 
the size of an egg, on the side of the neck, where 
inoculated. The diagnosis of no tuberculosis, as 
based upon failure of reaction to tuberculin, will be 
confirmed or disproved by an autopsy later. These 
experiments were kindly made for me by Dr. 
Schroeder. 
So far as I have been able to find, the first note 
of successful cultivation of the tubercle-bacillus 
upon liquids free from albuminoid matter was made by myself in the New \ork Medical Journal of 
March, 1893. Since then Uchinsky, Proskauer, 
and others have used similar media. None has, 
however, reported a better growth than that obtained 
by a slightly modified form of the original culture- 
medium. It required some time until I could per- 
suade the germ to grow in this liquid, but after 
considerable coaxing it will now multiply as well 
here as on glycerin-peptonized beef-broth, the 
fourth generation having become accustomed to the 
change of food. 
The formula that I have found eminently adapted 
and used may be repeated here: 
4 
Water ....... 1000 cc. 
Glycerin ....... 70 grams 
Acid pot. phos. ...... 1 gram 
Ammonium phos. ..... 10 grams 
Sodium chlorid 10 “ 
Asparagin 3 “ 
Magnesium Sulphate 0.2 gram 
After a certain amount of growth has appeared 
in a definite volume of media the growth will en- 
tirely cease. The germ has exhausted the supply of 
nutritive material or eliminated enough poison to 
check its own development, and, although the germs 
may still float on the surface of the liquid, after from 
three to four months there is practically no growth in 
the liquid. Further, there is a distinct and peculiar 
odor generated in the culture-media. This is due 
to a fatty acid which is difficultly volatile with 
steam. The reaction of the media when inoculated 
is slightly alkaline. As the growth increases it be- 
comes less so, and finally it is acid in reaction. 5 
In order to see if the apparent loss of virulence 
of the germ might be due to some of its products 
contained in the body of the germ, two guinea-pigs 
were inoculated with the twenty-third generation of 
the germ, after the germs had been carefully and 
thoroughly washed with sterile water to remove all 
products. These pigs, after two months, are still 
well. The bacillus grown on purely artificial media 
might naturally be supposed to be still more at- 
tenuated than that grown on the ordinary culture- 
liquid. To test this, some of these germs were used 
to inoculate guinea-pigs, with the same results as 
those already noted. 
In the case of a disease like tuberculosis, in 
which the incubation-period is often an exceed- 
ingly long one, there is a possibility that what now 
appear immune animals may in time succumb. In 
view of the fact, however, that all of the checks 
died so promptly, so long a time has elapsed since, 
and that all the other animals remained well, it is 
fair to presume that we have here a true immunity 
secured by an attenuated germ. 
The following notes give the experiments in de- 
tail, and in the table they can be noted in a com- 
prehensive form. 
The autopsies are not given in full, as they ap- 
peared superfluous. 
January 18, 1894. Guinea-pigs, Nos. 27, 28, 29, 
and 30, about three-quarters pound weight each, were 
inoculated with an emulsion of an agar-culture of 
tuberculosis, receiving 0.25 c.c. each, in the thigh. 
This was the eighteenth generation of the particular 
germ. On February 13, 1894, No. 27 was used to 6 
test some tuberculin, and gave a good reaction, 
although apparently in good health. February 15, 
1894, No. 28 was found dead, but the autopsy did 
not reveal any signs of tuberculosis. No. 27 was 
found dead on March 19th, two days after it had 
been fed a large amount of tuberculin. The autopsy 
did not reveal the presence of any tuberculous lesions. 
Nos. 29 and 30 were subsequently, within six months, 
chloroformed and examined, but no signs of tuber- 
culosis were apparent. 
February 16, 1894. Healthy guinea-pigs, Nos. 
115, 113, 128, and 127, were fed varying doses of 
tuberculin, from 3 c.c. to 12 c.c. Nos. 127 and 128 
succumbed to the feeding, but this proved to be due 
to the glycerin with which the tuberculin had been 
mixed. 
On March 30, 1894, Nos. 114, 115, T13, 109, 
and 112 were inoculated with 0.1 c.c. of tubercu- 
losis emulsion from the seventeenth generation of a 
liquid culture. June 5, 1894, No. 115 was chloro- 
formed, but the autopsy did not reveal any signs of 
the disease. October 1, 1894, No. 114 was found 
dead outside of the cage; it had been killed by the 
rats and the head was badly eaten. The animal was 
in good condition and fat, and the autopsy did not 
reveal any signs of tuberculosis. There was a little 
thickening of the skin and a small non-tuberculous 
lump at the point of inoculation. 
During April and May, Nos. 113 and 112 were 
fed on three different dates with small doses of 
tuberculin, but did not show any reaction. Both 
of these pigs are apparently in good health and en- 
tirely well at the present writing, eight months after 
inoculation. During this time guinea-pig No. 109 
had been chloroformed, September 29, 1894, and 
examined without any signs of disease being appar- 
ent. The seventeenth generation of this germ was 
evidently too attenuated to cause death. 7 
April 4, 1894. Guinea-pig No. no was fed with 
9 c.c. tuberculin, diluted with sterile water without 
ill effect. 
February 19, 1894, pig No. 122 was inoculated 
with 0.25 c.c. of tuberculosis culture, the seven- 
teenth generation, and on April 4, 1894, was fed 
with 25 c.c. tuberculin. The pig was rather thin; 
there was a lesion the size of a pea at the point of 
inoculation, but the temperature was normal. April 
9, 1894, this pig, No. 122, was found dead. The 
organs were apparently normal, except that the 
bloodvessels of the stomach and intestines were much 
congested. The pig had evidently been poisoned 
with the tuberculin ; the contents of the intestines 
were thick and mucus-like. 
February 19, 1894. Guinea-pig No. 121, weight 
13 ounces, was inoculated with 0.25 c.c. tubercle 
culture. May 16th it was well and was reinoculated 
with 0.25 c.c. tubercle culture. 
February 19, 1894. Guinea-pig No. 118 was in- 
oculated with 0.25 c.c. of tubercle culture, the 
seventeenth generation. March 19th, this pig was 
found dead, but the autopsy did not reveal any 
signs of tuberculosis. 
February 19, 1894. Guinea-pig No. 119, weight 
14 ounces, received an injection of 0.2 c.c. tubercle 
culture, and on March 24, 1894, an injection of 
0.5 c.c. of tuberculin. The temperature-reaction 
was very slight. 
February 19, 1894. Guinea-pig No. 120, weight 
14 ounces, was inoculated with 0.2 c.c. tubercle 
culture. On April 4, 1894, it was fed 3 c.c. of 
tuberculin. At this time it appeared to be ill, but 
did not show any reaction to the tuberculin, and 
the next day was quite well. 
February 19, 1894. Guinea-pig No. 125 was in- 
oculated with 0.25 c.c. tubercle culture. March  
Inoculated with 
Reinocu- 
Fed tuberculin. 
Injected tuber- 
culin. 
artificial culture 
attenuated 
Fed tuberculin. 
Injected tuber- 
culin. 
tion with 
tub.gland. 
lated with 
attenuat’d 
Dead. 
Chloro- 
formed. 
Autopsy. 
Present condition 
tubercle bacilli. 
culture. 
No. of animal. 
Amount 
Date. 
Am’nt. 
Date. 
Am’nt. 
Date. 
and gen- 
Date 
Am’nt. 
Date. 
Am’nt. 
Date. 
Daie. 
Date. 
Date. 
Date. 
Condition. 
Date of appearance, Nov. 28, 1894. 
1894 
.1894 
1894 
eration. 
1894 
1894 
1894 
1894 
l894 
1894 
1894 
27 
Jan. 18 
V c.c. 
Mar. 17 
24 c.c. 
Feb.13 
V c.c. 
Mar 19 
Mar. 19 
Poisoned with 
tSth gen. 
tuberculin ; 
no tubercu- 
losis. 
28 
“ 18 
* * 
Feb. 15 
29 
“ 18 
“ 
Mar. 19 
Organs nor- 
mal. 
3° 
“ 18 
“ 
“5 
II3 
no 
Feb.16 
“ 15 
April 4 
to c.c. 
4 c.c. 
9 c.c. 
Mar.30 
“ 3° 
c.c. 
17th gen. 
June 5 
June 5 
Well, fat, and in good condition. 
lulv 2Q 
Oct. 29 
Oct. 29 
Badly tuber- 
culous. 
141 
Mar. 8 
18 c.c. 
May 16 
July 29 
Well, fat, and in good condition. 
20th gen. 
142 
“ 8 
26 c.c. 
April 11 
April 11 
Tuberculin- 
136 
20th gen. 
poisoning. 
12 C.C. 
“ l6 
July 2q 
Oct. 1 
Oct. 1 
Organs nor- 
mal. 
135 
I37 
<< 
Well, fat, and in good condition. 
“ l6 
it 
20th gen. 
pigs ' 
2I3l 
Sept. 11 
Sept. 10 
Badly tuber- 
culous. 
214 j- 
Checks 
i% 
Sept. 11 
Sept. 11 
215 
Mar.24 
Sept. 21 
Sept. 21 
“ 
*33 J 
Vi c.c. 
July 27 
“ 
Sept. 17 
Sept 17 
“ 
y c.c. 
22d gen. 
Well, fat, and in good condition. 
“ 27 
Feb. 19 
Well, fat, and in good condition 
125 
Mar.24 
X c-c• 
19th gen. 
122 
April 4 
25 c.c. 
April 9 
April 9 
Tuberculin- 
poisoning. 
May 16 
V cc. 
Well, fat, and in good condition. 
17th gen. 
0.2 C.C. 
April 4 
3 c c 
Well, fat, and in good condition. 
17th gen. 
119 
Mar.24 
y c.c. 
Well, fat, and in good condition. 
Il8 
1 
Vi, C.C. 
Mar. 19 
Mar. 19 
Organs nor- 
Mar.30 
17th gen. 
mal. 
"4 
“ 
Oct. 19 
Killed by 
Oct. 1 
No tubercu- 
rats. 
losis. 
109 
<f 
“ 
Sept. 29 
Sept. 29 
‘ * 
Well, fat, and in good condition. 
Well. 
Rabbit 
Mi 
Feb. 23 
17th gen. 
July 27 
V\ c.c. 
20th gen. 
Cow 
217 
July 
20th gen. 
in the 
Tested with tuberculin; no re- 
action November 20, 1894. 
vein. 
Table Showing Effect of Attenuated Tubercle Cultures and.. Feeding Tuberculin in Guinea-pigs, Rabbit, and Cow. 10 
24th it received an injection of a small quantity of 
tuberculin, which caused a characteristic reaction. 
March 8, 1894. Guinea-pig No. 141, three-quar- 
ters of a pound in weight, was given 18 c.c. of dilute 
tuberculin. 
March 8, 1894. Guinea-pig No. 142, healthy, was 
fed 27 c.c. of tuberculin, but did not show any re- 
action. April 11, 1894, this pig was dead; the 
lungs were hemorrhagic, and the bloodvessels of the 
stomach and intestines much congested from tuber- 
culin-poisoning. 
March 12, 1894. Guinea-pig No. 136, healthy, 
was fed 12 c.c. of tuberculin. 
May 16, 1894. Guinea-pig No. 135, weight one 
pound; guinea-pig No. 141, weight one pound; 
and guinea-pig No. 136, weight one pound, all 
received 0.25 c.c. tubercle culture, the twentieth 
generation. Guinea-pig No. 137, weight one pound, 
received 0.5 c.c. of the twentieth generation of 
tubercle culture. Nos. 141 and 136 had both been 
fed tuberculin once, the others had never been used. 
July 27, 1894. Guinea-pigs Nos. 210 and 211, 
weight one pound each, received 0.5 c.c. of atten- 
uated culture of tuberculosis, the twenty-second 
generation. At the present writing neither of these 
animals shows the least signs of tuberculosis. 
July 29, 1894. Pigs Nos. 135, 136, 120, no, 
141, 125, and checks, healthy pigs, Nos. 213, 214, 
215, and 133, were all inoculated for me at my 
request by Dr. V. A. Moore, with the material from 
the tuberculous gland of a badly-diseased cow, No. 
286. The inoculations were made beneath the skin 
of the side. On September 11 th checks Nos. 214 and 
215 were both found dead, and the autopsy revealed 
extensive tuberculosis in lungs, liver, and spleen. 
September 21st, check No. 213 was found dead, the 
autopsy showing advanced tuberculosis. September 11 
17th, guinea-pig No. 133 was chloroformed for ex- 
amination, which revealed extensive tuberculosis. 
October 13, 1894. Guinea-pig No. 136, one that 
had been inoculated in May with the attenuated 
culture, and subsequently inoculated with a tuber- 
culous gland, was chloroformed for examination. 
Not a sign of disease could be detected and the 
local lesion from the inoculation had almost healed. 
October 29, 1894. Guinea-pig No. no was found 
dead. This was a pig that had been fed upon tuber- 
culin on April 4, 1894, and inoculated with the 
gland July 29, 1894. The autopsy showed advanced 
tuberculosis. The other pigs that had been inocu- 
lated with the attenuated culture and then the active 
virus, Nos. 135, 120, 141, and 125, are, at the present 
writing, quite well, and show no signs of disease. 
I may find that some of the animals that are now 
considered immune will eventually succumb to the 
disease. This, however, does not appear probable, 
as pig No. 136, examined after the death of all the 
checks, did not show any signs of disease. 
Further, animals No. 137, inoculated with the 
twentieth generation May 16th, and No. 112, inocu- 
lated March 30th; Nos. 120 and 119, inoculated 
February 19th ; No. 113, March 30th ; and No. 121 
on February 19th, and again on May 16th, are, at 
this writing, to all appearances perfectly well, having 
lived from six to nine months after the inoculation. 
February 23d, rabbit No. 141 was inoculated with 
0.25 c.c. of tubercle culture of the seventeenth gen- 
eration. July 27th, this same rabbit was reinoculated 
with the twentieth generation of a tubercle culture, 
0.25 c.c. being used. Still the animal is apparently 
well. 
These experiments which, of course, in connection 
with so important and troublesome a disease, can be 12 
regarded as preliminary only, and must be con- 
firmed by a large number of similar ones, serve to 
show that by the use of attenuated tubercle-bacilli 
and their products we may be able to control pul- 
monary tuberculosis. If the twenty-second genera- 
tion has shown such attenuation and produced so 
much resistance, then the thirtieth generation, or 
later, should give still more satisfactory results. 
An examination of the table and accompanying 
notes will also show that tuberculin, when fed in very 
large doses to supposedly diseased or to healthy pigs, 
may cause the death of these animals by poisoning. 
The amount necessary varies from twelve to twenty 
times the quantity that would give a reaction in a 
tuberculous cow. 
The digestion-experiments referred to in my re- 
port, in Bulletin No. y, Bureau of Animal Industry, 
Department of Agriculture, have shown that the 
action of tuberculin upon diseased animals is not 
affected after twenty-four hours in a hydrochloric- 
acid pepsin-solution, nor by warming with acetic 
acid. On the other hand, in two cases noted, feed- 
ing the tuberculin in small doses, while it did not 
prevent the disease, apparently retarded it. Possibly 
a combined feeding of tuberculin and the inocula- 
tion with the attenuated germ may be found more 
useful than either alone. 
The fact must not be lost sight of that the num- 
ber of bacilli injected into an animal may cause 
great differences in the length of time of the devel- 
opment of the disease. There could be no error of 
this sort, however, in the experiments recorded, as 
the emulsion of virulent and attenuated cultures 13 
used for inoculation were prepared in the same way, 
and a larger quantity of the attenuated culture was 
used than is necessary in case of a more virulent 
one to produce death, in five to six weeks. 
What conclusions may be drawn from these ex- 
periments? We have guinea-pigs made apparently 
distinctly immune to an active tuberculous virus 
by the use of an attenuated germ. We have guinea- 
pigs in which a single injection of a small quantity 
of tuberculin increased, perhaps, the subsequent 
immunity secured by injection with the attenuated 
culture. We have other pigs the immunity of which 
has not been tested, it is true, by an active virus, 
but which were inoculated with the attenuated cul- 
ture many months ago, and are still well, and in 
which an autopsy of other animals treated in exactly 
the same way and at the same time showed no signs 
of disease. 
Recently Viquerat claims to have been able by 
means of subcutaneous injections of the serum of the 
ass, an animal naturally immune to tuberculosis, to 
effect cures and improvement in the disease in man. 
Our attenuated germ may possibly prove very 
valuable in checking or controlling tuberculosis in 
animals, especially cattle. The Medical News. 
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