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IMMUNOLOGICALSTUDIESON PUREVARIOUS SPIROCHETES.

CULTURESOF

BY HIDEYO NOGUCHI, /vLD., Am) SEINAI AKATSU, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, December 15, 1916.)
INTRODUCTION.

As a sequel to the successful cultivation of Treponema pallidum and otherallied forms of spirochetes, ~ an era of test-tube experimentation on the questionof immunity in syphilis has been inaugurated. Previous to the time when theseorganisms were obtained in pure cultures, extensive experiments were carriedout with suitable animals by various investigators, notably by Metchnikoff andRoux, 2 Neisser and his associates, 8 Landsteiner and Finger, 4 Uhlenhuth andMulzer, 5 and others. Summing up their results, we are confronted with the factthat in syphilis no immunity, in the sense generally understood in bacterial in
fections, is demonstrable either in animals or in man. s,7 The insusceptibilityof animals or human beings who have once contracted the infection and havesince been apparently cured of a subsequent infection with Treponema pallidum,a fact well recognized since the time of Ricord, s is no longer regarded as a state of
immunity, but of an anergy, 3 which means that the same organism is not capableof responding to another infection as long as there is a preexisting infection.
Moreover, clinical and experimental data show that when an individual is curedof an attack, he soon regains practically his original susceptibility to a second
1Noguchi, H., Mi~nch. meal. Woch., 1911, lviii, 1550; J. Exp. Med., 1911, xiv,
99; 1912, xv, 81, 90, 466; xvi, 194.
Metchnikoff, E., and Roux, E., Ann. Inst. Pasteur, 1903, xvii, 809; 1904,
xviii, 1, 657; 1905, xix, 673; 1906, xx, 785.
3 Neisser, A., Beitriige zur Pathologie und Therapie der Syphilis, Berlin, 1911;
also Arb. k. Gsndhtsamte., 1911, xxxvii, 569; Deutsch. reed. Woch., 1006, xxxii, 1,
97.
4 Landsteiner and Finger, Centr. Bakteriol., 1re Abt., Ref., 1906, xxxviii, Bell.,107. Landsteiner, K., Centr. Bakteriol., 1re Abt., Ref., 1908, xli, 785.
5 Uhlenhuth, P., and Mulzer, P., Arb. k. Gsndhtsamte., 1913, xliv, 307.
e Zinsser, H., J. Lab. and Clin. Med., 1916, i, 785.
7 Levaditi, C., Z. Immunit~tsforsch., Ref., 1910, ii, 277-318.
s Ricord, P., Trait6 de la syphilis, Paris, 1845.
765

766

IMMUNOLOGICAL STUDIES O N SPIROCHETES

infection. 9 In this respect, syphilis bears more resemblance to a protozoan than
to a bacterial disease. Attempts to study the immunity question in vitro havenot been lacking, and numerous investigators claim to have observed diverse
specific immunity reactions, such as precipitation, TM immobilization,lz complement fixation, ~' 13 etc., when the serum or cerebrospinal fluid had been broughtin contact with a material containing the treponemata of syphilis. But analysisof these observations proved that many of these phenomena could not be confirmed, and none was strictly specific. The Wassermann reaction is very constantin syphilis, but not specific. I t is evident, therefore, that there is no recognizedspecific antigen-antibody reaction in syphilis. SchereschewskyTM tried agglutination tests with his impure cultures of a spirochete derived from syphilitic material, with inconclusive results. We cannot, however, accept the negative findingsof earlier investigators as final until it has been demonstrated that the techniques employed cannot be further improved. The great obstacle in the way ofsatisfactorily testing immune reactions in syphilis lies in the fact that a sufficientquantity of virulent organisms free from tissue constituents is obtained onlywith difficulty. A pure culture would fill this requirement.
In 1912 Noguchi 15 had already commenced to employ his several strains ofculture pallidum for the purpose of studying the various immunity problems
which were awaiting solution by means of a culture material. The main efforts
were directed to finding out whether a rabbit repeatedly inoculated with the pure
pallidurn cultures will become resistant to a subsequent inoculation with a virulent
pallidum. The cultures used were already avirulent. For comparison, anotherseries of rabbits was similarly treated with live and killed virulent pallida forthe same length of time, which covered a period of 5 months. The mode of immunization consisted in intravenous and intratesticular inoculation, except in
the case of the live virulent material, which could not be used intratesticulariyon account of its tendency to start the infection in the organ. The aim of theintratesticular mode of immunization was to find out whether there is such athing as a local immunity in syphilis. The immunized animals were tested witha virulent strain by inoculating it into their testes. I t was found that six out of
the twelve rabbits immunized with the culture pcdlidum intravenously took the
inoculation, while five of the twelve rabbits receiving the virulent pallidum took.
Those which were immunized with the pallidum emulsions (live culture and killed
9 John, F., Samml. klin. Vortr., 1909, 559 ft.
10 Fornet, W., Berl. klin. Woch., 1908, xlv, 85.
11 Hoffmann, E., and von Prowazek, S., Centr. Bakteriol., lie Abt., Orig., 1906,
xli, 741,817.
12Detre, L., Wien. klin. Woch., 1906, xix, 619.
J3Wassermann, A., Neisser, A., and Bruck, C., Deutsch. reed. Woch., 1906,
xxxii, 745.
14 Schereschewsky, Deutsch. reed. Woch., 1909, xxxv, 1652.
J5 Noguchi, J. Am. Med. Assn., 1912, lv~ii, 1163.

]fflDEYO I~0GUCBI A N D

SEINAI A K A T S U

767virulent strains) lost much of the glandular structure of their testes, some becoming mere strands of hard connective tissue. Positive takes were recordedin three of the six rabbits treated with the culture and two of the six treated withthe killed pallidum from a syphilitic orchitis of the rabbit. As the number ofanimals was so small it was impossible to draw any conclusion. The results ascompared with control series with normal rabbits were striking, since the takes
in the normal animals were practically 90 per cent. There was an indicationthat the repeated inoculation of the rabbit with the pallidum material reduced
susceptibility to a certain extent, although the difference may have been due tothe altered structure of the inoculated testes. The immune sera obtained fromthese rabbits were aiso tested for agglutination, spirochetolysis, complement
deviation, and opsonic property against their homologous and cross antigens.
The results were at first encouraging, but repeated experiments soon showed
them to be indecisive on account of the technical difficulties in the way of obtaining satisfactory antigens. This was so even with the culture pallidum,
which at that period was either very difficult to obtain free of culture media or
underwent spontaneous agglomeration.
Craig and Nichols, 16 employing alcoholic extracts of pure cultures of thepallidum, pertenuis, and microdentium, furnished by Noguchi, reported that theseantigens fixed complement with syphilitic sera very much as does an alcoholic
extract of a congenitally luetic fetal liver.
Kolmer, 17 in the meanwhile, employing a strain of culture pallidum furnishedby Noguchi, prepared immune sera in the rabbit and demonstrated the presenceof agglutinins for the strain used. His most powerful serum agglutinated thepallidum in a dilution of 1:1,280. Kolmer, Williams, and Laubaugh is next
studied a series of human and animal sera with regard to complement fixation,with the culture pallidum as antigen. Their positive findings, although more
numerous, were nevertheless similar to those already obtained by Noguchi. ]5On the other hand, they noticed that rabbit immune sera not only fixed complement with their homologous antigens, but also with washed typhoid and cholera
antigens. They point out, as was previously emphasized by Noguchi, 19 thatthese immune sera do not fix complement with the alcoholic extract of the culture

pallidum.
Kissmeyer,2° employing a strain of culture pallidum as antigen in the agglutination tests with human sera, obtained a consistent result when compared withthe clinical and Wassermann reactions, but this apparent specificity was evidently
t6 Craig, C. F., and Nichols, H. J., f. Exp. Med., 1912, xvi, 336.
t7 Kolmer, J. A., J. Exp. Med., 1913, xviii, 18.
18 Kolmer, J. A., Williams, W. W., and Laubaugh, E. E., f. Med. Research,
1913, xxviii, 345.
1~ Noguchi, J. Exp. Med., 1909, xi, 84.
20 Kissmeyer, A., Deutsch. reed. Woch., 1915, xli, 306.

768

YMM-UNOLOGICALSTUDIESONSPIROCHETES

not confirmed by Zinsser, Hopkins, and McBurney. 21 The recent contributions
made by these investigators2~ have advanced our knowledge of the immunity
phenomena in connection with the cultivated avirulent and uncultivated virulent pallidum strains; comparing the behavior of both towards the immune sera
prepared by means of the former. They came to the conclusion that the immune serum behaves in the same manner as a bacterial immune serum whentested upon the culture pallidum; that is, it produces agglutination, causes destruction of the organism with the cooperation of fresh complement, and fixes
complement with the culture antigen. On the other hand, no immunity phenomena are demonstrated when the immune serum and the virulent uncultivatedorganisms of the same strain are brought together. They have not mentioned,
however, whether or not an immune serum prepared by means of injecting thevirulent material repeatedly will ever produce a protective or spirocheticidat
principle.
The work of Kolmer and Zinsser, Hopkins, and McBurney, to which we havereferred, has brought out a number of facts, particularly with regard to the relation between the avirulent culture and the virulent tissue pallidum strains, but
the question is still far from being solved. It is not superfluous, therefore, thatany data bearing upon this phase of the study should be published.
T h e p r e s e n t p a p e r deals with the results of immunological studies
which h a d been i n t e r r u p t e d b u t were recently resumed. As will beseen f r o m the following account, we h a v e used a considerably largern u m b e r of culture spirochetes a n d of animals a n d continued the imm u n i z a t i o n longer t h a n a n y worker has previously reported. W e
h a v e p u r p o s e l y avoided touching the question of the relation between the cultivated a n d u n c u l t i v a t e d strains, as we expect to consider t h a t p o i n t in a future paper.
EXPERIMENTAL.

Material and Scope of Experiments.
I n the p r e s e n t series of i m m u n i z a t i o n experiments, r a b b i t s of a b o u t
3 kilos were employed. Several r a b b i t s were used for each of the four
strains of Treponema pallidum, a n d several also for each of the following: Treponema calligyrum, Spirochceta refringens, Treponema
microdentium, and Treponema mucosum. T h e m o d e of i m m u n i z a t i o n
21Zinsser, H., Hopkins, J. G., and McBurney, M., J. Exp. Med., 1916, xxiv, 561.
22Zinsser, H., and Hopkins, J. G., J. Exp. Med., 1915, xxi, 576; 1916, xxlii,
323. Zinsser, Hopkins, and McBurney, ibid., 1916, xxiii, 341.

769

H I D E Y 0 N O G U C H I AND S E I N A I A K A T S U

consisted in the intravenous injection every week of 1.5 to 3 cc. of
the fluid culture of each strain, the strains having been grown in a
medium consisting of equal parts of normal rabbit serum and faintly
alkaline bouillon, with a piece of fresh rabbit kidney. The ascitic fluid
usually used was replaced by rabbit serum in order to avoid the production of a specific precipitin or of complement-binding antibodies
for human proteins in the animals immunized against the spirochetal
cultures. The organisms grew fairly well in this medium, owing
undoubtedly to their gradual adaptation previously to various culture media which originally were unsuitable for their growth. The
age of the cultures varied from 14 to 30 days, and the number ofspirochetes was approximately thirty to forty per field when exam1
ined under the dark-field microscope (Leitz oc. 3, ~ oil immersion).
The aim of these immunization experiments was to study thedevelopment of such agglutinins, complement-binding antibodies,
opsonins, and spirocheticidal principles as may be demonstrated invitro in the sera of rabbits repeatedly inoculated with spirochetes.
The immunity reactions were first tested with the specific organisms
and then with those not used for the production of the immune sera
in question. The cross examinations, various non-specific as well as
T A B L E I.
Immunization

Rabbit No.

Tests

with

Treponema

pallidum.

T. ~allidum.

Period of immunization.

No. of injections.

Strain M c D .

Sept. 14-Dec. 17" 14- " 17
" 10- " 17Oct. 2 0 - " 17

13
13
11
8

Strain XI.
" XI.

Sept. 14-Dec. 17"
14- " 17
"
30- "
17
"
3 0 - " 17

13
13
11
11

1915

5
6
7

"

XL

8

"

XI.

9
10

Strain B29.
"
B29.

Sept. 14-Dec. 17
"
30- "
17

13
11

11
12

Strain Z . A .

Sept. 14-Dec. 17"
14- " 17

13
13

"

"

770

IMMUNOLOGICAL STUDIES ON SPIROCHETESt h e specific a n t i g e n s being used a g a i n s t a given i m m u n e serum, wereu n d e r t a k e n w i t h a view t o establishing t h e relation w h i c h m a y existb e t w e e n different m e m b e r s of t h e g r o u p of spirochetes.
T h e protocols of i m m u n i z a t i o n are given in T a b l e I.
T h e results of e x p e r i m e n t s w i t h t h e i m m u n e sera m a y be s u m m a r ized u n d e r s e p a r a t e heads.

A gglutinins.
T h e m o d e of d e t e r m i n i n g t h e c o n t e n t of agglutinins in each s e r u m
w a s c o n d u c t e d in t h e following m a n n e r .
Into a series of small sterile test-tubes were measured quantities of the immune
serum in amounts graduated from 0.1 to 0.000001 cc. for each tube. The distribution of the various amounts was carried out as is usual in such procedures;
namely, by adequately diluting the serum with a 0.9 per cent saline solution andthen taking out such quantities of each dilution as are required for titration.
To each of the tubes containing various quantities of the serum was added 0.1
cc. of the spirochetal emulsion (as antigen), and the final volume was brought up
to 1.5 cc. by adding the necessary quantities of 0.9 per cent saline solution toeach tube. The content of the tubes was thoroughly mixed by shaking and thetubes were placed in a water incubator at 37°C. At least two tubes containing
the spirochetal emulsion alone were prepared at the same time and served ascontrols. Concentrated and washed suspensions of various spirochetes derived
from pure cultures in a fluid medium were used as antigens. 0.1 cc. of thesuspension of each strain in 0.9 per cent saline solution was used for eachtube. The number of spirochetes varied in different suspensions, but there
were over 100 per field (Leitz oc. 3, ~ oil immersion, dark-field illumination),and the addition of 0.1 cc. of the antigen emulsion to each tube (total volume of
fluid, 1.5 cc.) produced grayish white, semitranslucent turbidity. The turbidity
in the control tubes gradually subsided while standing in the incubator, but wasnever completely cleared up, even after 24 hours' standing. Readings of theresults were made twice, once after 2 hours' incubation, and again at the end of
24 hours at a temperature of 15°C. The sera were used without any modification, such as inactivation at 56°C.
As m a y be seen f r o m T a b l e I I , we first t e s t e d t h e a g g l u t i n a t i n g
p o w e r s of v a r i o u s i m m u n e sera for their h o m o l o g o u s strains; t h a t is,for t h e strains w h i c h h a d b e e n u s e d for p r o d u c i n g t h e m in t h e r a b b i t s
b y r e p e a t e d i n t r a v e n o u s injections. T h u s , eleven sera were t i t r a t e d
w i t h four different strains of t h e pallidum, t w o t o four i m m u n e
sera for each strain. As in t h e case of the pallidum t h e r e m a i n i n g

HIDEYO NOGUCHI AND S E I N A I AKATSU

771

immune sera, eight in number, were tested with their homologous
species, the calligyrum, refringens, microdentium, and mucosum, which
had been employed for their production.
It m a y be mentioned that we had to make a number of preliminary
experiments before we felt assured of obtaining fairly uniform andreliable results. The reading of a strong agglutination was quite
easy, as the organisms rapidly settled down, and the sediment adhered firmly to the side and bottom of the tube, but the less intense
reaction was not as clear as we wished; hence, our reading of theminimum zone of the reaction was more or less arbitrary. The titersof agglutinins in different sera read after 2 hours' incubation were
somewhat lower than those recorded after the same set of tubes had
been left at room temperature for 24 hours longer. It was always
necessary, in order to detect a slight degree of agglutination, to shakeup the sediment and compare the granulation or clumping of the suspension with a control tube without any serum. We have resorted
solely to the macroscopic reading of the reaction. A microscopic
examination of the sediment adhering rather firmly to the side andbottom of the test-tubes where a definite agglutination occurred revealed enormous masses of entangled spirochetes, some apparently
undergoing morphological modifications, as shown by a granularappearance or by relaxation of curves.
In Table II several facts seem to stand out conspicuously. In thefirst place, the titers of agglutinins developed in the rabbits treatedwith the saprophytic species, namely, the calligyrum, refringens,microdentium, and mucosum, are decidedly lower than those foundin the sera produced by immunizing the rabbits with the pallidum.
Secondly, the amounts of agglutinins in different immune sera are
not in direct proportion to the number of injections or the durationof the immunization. Thus, the highest titer was found in Serum 4,in which 0.00001 cc. caused a definite, and 0.000025 cc. a slight agglutination, while Serum 9, notwithstanding the fact that the animal had
five more injections than the former, required 0.0025 cc. for a definite
reaction. Again, it is evident, with regard to the production of theagglutirdns, that different rabbits react differently to the same
spirochetal antigens. The apparent difference between the titersof the pallidum group and those of the other group requi~es an ex-

772

IMMUNOLOGICAL STUDIES ON S P I R O C H E T E S

TA B LE I I .

~Fubes Incubated for 2 Hours in the Water Bath. Antigen: 0.1 Cc. of Emulsion of
F l u i d Cultures 2 Months Old. Total Volume 1.5 Cc.
Antigens.

Immune sera.

TJters of agglutinins.

No. 2

O.000025
0.0001O.00000375
0.00005
0.00050.00004
O.000025
0.001
0.00005
0.001
0.0OOO25
O.00050.001
0.001250.001
0.0025
0.001
0.001
0.01

T. pallidum,
Strain"""McD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
XI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Z. A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T. calligyrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
,S. refringens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
T. microdentium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
T . ~fgc, osuf'n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

"

3

~c

4

u

5

~c

6

u
~c

7
8

cc

9

10
11
12
13
" 14
" 15
" 17
" 18
" 19
" 20
" 21
"
"
"
"

planation. W h e t h e r t h e p h e n o m e n o n is due to the greater a m o u n t sof agglutinins produced in the r a b b i t s i m m u n i z e d with the p a l l i d u mor to a physical or possibly also chemical factor inherent in the p a l l i d u m antigen has n o t been established.
I t is also possible t h a t our
results with the s a p r o p h y t i c varieties h a p p e n e d to be inferior m e r e l ybecause the animals used were u n f a v o r a b l e individuals. T h e resultsm i g h t h a v e been different if we h a d e m p l o y e d m o r e animals.
Of m o r e interest are the results of cross exazninations of various
i m m u n e sera with regard to their specificity t o w a r d s the homologous
a n d heterologous antigens. E i g h t i m m u n e sera were chosen to betested, each with six strains of T r e p o n e m a p a l l i d u m a n d one of T r e p onema calligyrum, Spirochceta refringens, Treponema microdentium, andT r e p o n e m a m u c o s u m , or ten different antigens in all. Stated in m o r edetail, the procedure was as follows:HIDEYO NOGUCHI AND SEINAI AKATSU

773

Serum 4 was produced by injecting the animal with Strain McD. of the pallidurn and was tested not only with the emulsion of the same strain (homologous),but also with those of the Strains XI, R, C,, B30, and Z. A. of the pallidum, as
well as With those of the calligyrurn, refringens, rnicrodentium and mucosum(heterologous and of different species). Serum 8, produced with Strain XI, andSerum 12, produced with Strain Z. A. of the pallidum, were likewise tested against
their homologous as well as their heterologous antigens, while Serum 10, thehomologous antigen of which was Strain B29, was tested with all ten heterologous
antigens, since there was no homologous one in the series. Sera 14 and 17 of
the calligyrum and refringens of the non-pathogenic spirochetes of the genitalia,
and Sera 18 and 20 of the microdentium and mucosum of the buccal cavity were
each tested with one homologous and nine heterologous antigens. In order tosecure as closely comparable results as possible, all the tests were carried out inparallel series on the same day and with the same materials. The first reading
was recorded after completion of the 2 hour incubation in the water thermostat
and the second at the end of the 24 hour period at room temperature. The
latter readings are given in Table III.
I n the analysis of Table I I I , several points are brought out.
I n the first place, there is a pronounced degree of specificity ofeach characteristic for its homologous group of antigens. Thus,
the immune sera belonging to the paltidum group agglutinated moststrongly when brought together with the emulsion of the pallidum
strains, b u t not at all when mixed with the microdentium or mucosum.
T h e reverse is also true; t h a t is, the immune serum produced b y meansof the microdentium agglutinated none of the other groups, except
for a slight reaction with the mucosum. T h e mucosum immune serum
showed its strongest action on the mucosum antigen, although there
was a more or less feeble reaction with some of the other spirochetes,
especially the microdentium. T h e relation between the calligyrum
and refringens seems v e r y close, considered from the point of viewof the agglutination reaction. T h e y did agglutinate m u t u a l l y tosuch an extent t h a t t h e y might be included in the same group. As
already mentioned above, t h e y showed no affinity w h a t e v e r for thegroup of the m o u t h spirochetes. On the other hand, there existed
a certain degree of so called group reaction between the pallidum andthe calligyrum groups. I t m a y be pointed out t h a t in spite of theclose relation between the calligyrum and the refringens, the immune
sera pertaining to the pallidum did not noticeably agglutinate therefringens, while the same sera invariably agglutinated the calligyrum

774

IMMUNOLOGICAL STUDIES ON SPIROCHETESTABLE I l l .

Specificity of I m m u n e Sera towards the Homologous and Heterologous Antigens.
Results of Incubation for 24 Hours at Room Temperature.
I
Antigens.

Immune sera.

No. 4
No. 8
No. 10No. 12
No. 14
No. 17
No. 18
No. 20homolo- homolo- homolo- homolo- homolo- homolo- homolo- homologous with gous with gous with gous with gous with gous with gous with gous with
StrainStrainStrainStrainT. calli- S. refrln- T. micro- T. raucoMeD.
XI.
B29.
Z. A.
gyrum,
gens.
dentium,
sum.
Cd.

de.

CC,

CC.

co.

CC.

CO.

Co.

T. pallidum.
Strain M c D . . . .

"XI .....

"R ......

"

C2...

"

B30 . . . . . .

"

Z. A

T. caltigyrum...
S. refringens . . . . . .
T. microdentium..
T. mucosum.

0.0005 i 0.0005 0.0005 0.0025 ~.05

Trace N o n e a t N o n e a t
at
0.05.
0.05.
0.05.
0.0005 i 0.0005 0.0005 0.005 E ) . 0 5
Trace N o n e a t N o n e a t
at
0.05.
0.05.
0.05.
0.0005 0.005
0.005 0.005 D.05
0.05
Noneat Traceat
0.05.
0.05.
0.0005 0.001
0.0005 0.001 0.01
Trace None a! Trace at
0.05.
0.05.
at
0.05.
0.0005 0.0025 0.0005 0.005 0.05
Trace None at Trace xt
0.05,
0.05.
at
0.05.
0.0005 0.0005 0.0005 0.0005 0.05
0.05
None at Trace at
0.05.
0.05.
0.05
0.05
0.025 0.025 0.0005 0.0075 None at Trace at
0.05.
0.05.
None at None at None at None at 0.005
0.0025 None at Trace al
0.05.
0.05.
0.05.
0.05.
0.05.
0.05.
None at None a t None at None at None at None. 0.001
0.01
o 05.
0.05.
0.05.
0.05.
0.05.
None at None at None at None at None at
"
0.05
0.001
0.05.
0.05.
0.05.
0.05.
0.05.

in doses between 0.05 and 0.025 cc., that is, in a dilution of 1 : 20 to1 : 40. This group reaction is, of course, far below the titers of these
sera for the pallidum antigens, in which they varied from 0.005(1: 200) to 0.0005 cc. (1: 2,000), and still further down to 0.00001 cc.
(1: 100,000)(Table II). Conversely, the calligyrum serum, which
agglutinated its own antigen in a dose of 0.0005 cc. (1 : 2,000) produceda group reaction with the pallidum emulsions in doses ranging fromHIDE~20 NOGUCHI AND SEINAI AKATSU

0.05 (1 : 20) to 0.01 cc. (1 : 100).

775

In this respect, the behavior of therefringens serum was somewhat similar to that of the calligyrum, as
it also produced a distinct agglutination with some of the pallidum antigens when employed in a concentration stronger than 1 : 20,
sometimes even in a dilution of 1: 40.
As to the results obtained among the paUidum antigens with thecorresponding and cross immune sera, it will be noticed that thereaction between the homologous antigens and immune sera are, asa rule, stronger than those which occurred when the former weremixed with the sera produced by the other strMns. On the other
hand, a serum which strongly agglutinates a certain strain or strains
may not necessarily agglutinate others, while the latter may yet be
the most readily and strongly agglutinated by another serum. In
other words, our present study indicates that the aggiutinin titersof these sera are variable according to the differences in the individualstrains of the same group. This phenomenon has long been recognized in the agglutination of various bacteria by their immune sera,
and apparently it holds good in the case of the spirochetes. The
complex composition of agglutinlns, such as partial aggiutinins ofdifferent affinities or still only scantily understood factors in agglutination reactions, is accountable for the intricacy of the so called
specific as well as group reactions.

Influence of Time upon the Agglutinins in Vitro and in Vivo.
The immune sera used in the foregoing experiments were preservedin a refrigerator at the temperature of 6°C. for 3 months and thenexamined for their strength. In the meantime, the rabbits immunized were not given any further injection of the spirochetal emulsions for the same length of time, and then their sera were drawn
for the titration of agglutinins. Table IV shows the titers of these
sera as compared with their original strength.
The rates with which the agglutinin contents of the immune sera
lost strength during the 3 months seem to be irregular and show noconstant proportion to the original titers of the sera. There is,however, a general tendency of the agglutinins to disappear fromthe serum more rapidly in vivo than in vitro. The titers of the ag-

776

IMMUNOLOGICALSTUDIES O NTABLESPIROCHETESIV.

Effect of Time upon Agglutinins

in Vitro and in Vivo.
Titer

Immune sera.
Original.

After 3 mos.
kept at 6°C.

After 3 mos.
in the animal,

0.000250.00050.00010.0001
0.01
0.01
0.01
0.01

0.001
0.001
0.001
0.001
0.1
0.1
<0.1
<0.I

CG.

No.

2, T . p a l l i d u m , Strain McD . . . . . . . . . . .

""8, T .
""
XI .............
10, T .
""
B29 . . . . . . . . . . .
12, T .
""
Z.A ............
14, T . calligyrun . . . . . . . . . . . . . . . . . . . . .
15, S . refringens . . . . . . . . . . . . . . . . . . . . . .
19, T . m i c r o d e n t i u m . . . . . . . . . . . . . . . . . . .

"20, T. ~COSUrtt ......................

"
"
"
"

0.O00025
0.000025
0.00OO5
0.000025
0.001
0.00125
0.001
0.001

glutinin in the sera derived from these rabbits at the end of 3 months
after discontinuation of immunization were reduced to about oneone hundredth in Nos. 14, 15, 19, 20; one-fortieth in Nos. 2, 8, 12;
and one-twentieth in No. 10, while the original sera when preservedin the refrigerator during the same period of time lost their strength
down to about one-tenth of the initial titers, except in No. 8, where
it was almost twice as much weakened as in the rest. It may therefore be assumed that during the first 3 months after the cessation of
immunization, the disappearance of the agglutinins for various
spirochetes was almost ten times as fast in the animal body as in the
test-tubes kept at a temperature of 6°C.

Complement Fixation.
The immune sera were also tested for their property of binding
complement when mixed with their homologous as well as heterologous antigens. The technique of carrying out the tests was thatusually employed in such experiments.
T h e i m m u n e sera were i n a c t i v a t e d a t 56°C. for half a n hour before use. T h eantigens were p r e p a r e d from the pure cultures of various spirochetes grown in a
fluid m e d i u m consisting of equal p a r t s of r a b b i t serum a n d slightly alkaline
bouillon, with a piece of normal r a b b i t kidney. T h e spirochetes were collectedfrom cultures which h a d been vigorously growing for 4 weeks. For t h e purposeof concentration a n d purification, the spirochetes were collected b y a prolonged

HIDEYO NOGUCB_I AND SEINAI AKATSU

777

centrifugation and washed again in 0.9 per cent saline solution. The sediment, resuspended in an adequate volume of 0.9 per cent saline solution, showing about
twenty to thirty spirochetes under dark-field illumination, was used as the antigen.
The relative quantities of different factors were as follows: The immune serum inquantities of from 0.1 to 0.0001 cc., complement 0.04 cc. (2 units), antigen 0.2 cc.,
sheep corpuscles 5 per cent, anti-sheep amboceptor 0.0002 cc. (corresponding to2 units), total volume made to 1.5 cc. with 0.9 per cent saline solution. The first incubation was for I hour (at 36°C. in the water bath), and the second also for 1 hour.
The quantities of immune serum and the spirochetal suspensions chosen for theabove experiments were previously tested and found to be not anticomplementaryin themselves. The results were recorded after allowing the tubes to stand for
2 hours longer at room temperature.
TABLE V.
Complement

Fixationwith

Antigens 0.2 cc.

Homologous

Antigens.

Immune sera.

Titers of fixation.

No. 2
" 3

0.005
0.002
0.005
0.005
0.1
0.005
0.008
0.007
0.003
0.002
0.0003
0.01
0.1
0.03
0.OO7
0.05
0.002
0.005

T. pallidum.

Strain McD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

""XI .....................................

B29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

"

4

,c

5

"

6

"

7

"

8

"

9

10
" 11
" 12
" 14
" 15
" 17
" 18
" 19
" 20
" 21
"

"

Z. A ....................................

T. ealligyrum ....................................
S. refringens ....................................
T. microdentium .................................
T. mucosum .....................................

T a b l e V shows t h a t t h e titers of t h e i m m u n e sera d e r i v e d f r o mdifferent r a b b i t s v a r y c o n s i d e r a b l y a c c o r d i n g t o t h e individual v a r i a t i o n s t h a t exist a m o n g t h e animals. F o r example, I m m u n e Sera
6 a n d 15 s h o w e d a n e x t r e m e l y low p o w e r for c o m p l e m e n t fixation,in spite of t h e fact t h a t t h e r a b b i t s in t h e s a m e groups, p a r t i c u ] a r l y

778

IMMUNOLOGICAL STUDIES ON S P IR O C H ETESin the Strain X I series, produced fairly high titers. The titers of
the pallidum group were in the main quite high, while the calligyrum
and microdentium remained much less developed. It is interesting
to note that of the two animals treated with the mucosum, both
responded with high titers. When compared with the agglutinin
titers of the same sera, the fixation titers are much lower but seemto run fairly parallel, in the sense that a serum with stronger agglutinating power had also a higher titer for complement fixation.
There were a few exceptions to this rule.
For determining the question of specificity of the complement
fixation reaction with the spirochetes a series of experiments was
performed in which a certain number of the immune sera were tested,each in turn, with the entire set of antigens. Table VI gives theresults. The technique used was the same as before.
Table VI shows that various immune sera fixed complement morestrongly with their corresponding antigens than with a heterologous one. Among the pallidum strains, the reaction is fairly
interchangeable, although some immune sera (Nos. 3 and 7) havecaused only a weak fixation with a certain antigen (Strain B29). The
calligyrum serum produced a marked fixation, not only with its own
antigen, but also with certain pallidum strains (Strains X I and Z. A.)
to such an extent that in a dilution of 1:30 no difference could be
distinguished between the calligyrum and these two pallidum strains.
In this connection, mention may be made of an analogous instance
where one of the pallidum immune sera (No. 3) fixed complementwith the calligyrum antigen distinctly in a dilution of 1:30. The
refringens serum fixed complement best with its own antigen, but also
feebly with all the other antigens except that of the mucosum. A
similar group reaction was evident with the calligyrum and mucosumsera. In spite of the presence of a certain amount of group reaction,
the specific character of the complement fixation phenomenon seems
to hold. At least, the differences between the pallidum, refringens,microdentium, and mucosum are sufficient for considering the reaction
specific. The catligyrum showed much affinity for the pallidum on
the one hand and for the refringens on the other.

HIDEYONOGUCHI

AND

TABLE

SEINAI

779

AKATSU

VI.

Complement Fixation with tteterologous Antigens.
Antigens used for cross titration.

T. pallidum

Immune sera.

T. calli- S. re- T. mic- T. r,tu.
gyrum, fringens, rodentiura, cosum.

strain.

McD.

XI.

] B29.

0.1
0.03
O.Ol

+-4++
+

+-4++
+

<+
--

++
++

-4<+

++

.

.

No. 7 h o m o l o g o u swith Strain XI.

O. 1
0.03
0.01

-4"4<++-4+-t+++<+--+-4-4<+

-4.
.

.
.

No. 10 h o m o l o g o u swith Strain B29.

0.1
0.03

++
++

-[-+
++

++
++

++
++

<+
.

.

0.01

++

+

++

++

.

.

~ o . 11 h o m o l o g o u swith Strain Z.A.

0.1
0.03
0.01

+
<+

++
+

++
+

--

< +

++
++
++

<+
.
.

~o. 14 h o m o l o g o u s
w i t h T. calligyrum.

0.1
0.03
0.01

< <+
++
-<+
.
.
.

<+
-.

+
<+

++
<+
< <+

~qo. 17 h o m o l o g o u s
w i t h S. refringens.

0.1
0.03
0.01

<+
.
.

<+
.

<+

~qo. 18 h o m o l o g o u s
w i t h T. microdentlum.

0.1
0.03
O. 01

-.
.

.
.

hlo. 21 h o m o l o g o u s

0.1

<+

[ <+

<+

<+

w i t h T. mucosum.

0.03

.

I.

.

.

.

o.ol

.

.

.

.

gO.

No. 3 h o m o l o g o u swith Strain McD.

Z.A.

I

--

+
.

.
.

.
--

.
-.
.

.
.

---

< <+

--

--

--

--

--

--

<+
---

i < <+
---

< <+
---

+
+
< <+ < <+
.
.
.

< <+
--

---

, < <+

--

++
++
<+

<+
---

< <+

--

<+

.
<+
.
.

---

.

.
.

.
.

.

.

.

.

.
.

.
.

--

<+
.
.

.

.

.

--

.
.

.

.

--

++

+ +

.

++

Spirocheticidal Properties.
I n order to d e t e r m i n e whether or not these i m m u n e sera h a v e
a destructive power u p o n the spirochetes in vitro, several series of tests
were carried out. Because of the p a r t p l a y e d b y c o m p l e m e n t in abacteriolytic process, our experiments were so a r r a n g e d t h a t in one
set the i m m u n e s e r u m was allowed to act alone, while in another,b o t h c o m p l e m e n t a n d i m m u n e serum were p u t together with the
spirochetes.

780

IMMUNOLOGICAL STUDIES ON SPIROCttETES

The immune sera were inactivated at 56°C. for 30 minutes before use, and the
spirochetes were' from recent fluid cultures and were very active. The amountof immune serum was graduated from 0.1 down to 0.00005 cc., while spirochetes
and complement were used in a quantity of 0.1 cc. each for each test-tube. The
volume of the resulting fluid in each tube was made uniformly 1.1 cc. by adding
0.9 per cent saline solution. After all the factors, namely, spirochetes, immune
serum, and complement, had been mixed, the tubes were placed in a water bathincubator at 37°C. for 1 hour, and then the whole contents were used for cultivation in the usual ascitic agar tissue medium. The results were recorded at theend of 2 or 3 weeks of incubation at 36°C. Normal rabbit serum was also tested
as control.
T h e results of experiments in which most of the antisera were
tested show t h a t the anti-pallidum Serum 12 suppressed the growthof its homologous strain Z. A. in a dose of 0.001 cc. in the presenceof complement, while scanty growth was observed in all the tubes
where there was no complement added to the immune serum. Normal rabbit serum had no appreciable effect on the growth of thisstrain. I t was noted also t h a t the n u m b e r of spirochetal co]onies
diminished almost proportionately with the gradual increase of thespecific immune sera, until, in quantities of serum beyond a certain
limit, no growth was observed. With the anfi-pallidum Serum 4,
and possibly also No. 8, the titer was 0.0003 cc. I t was not rare forthe anti-pallidum sera to attain the titer of 0.001 cc. On the other
hand, the immune sera for the refringens, calligyrum, and mucosum
were far inferior in their restraining influence upon their homologous
species, none of t h e m being strong enough to inhibit the growth ina dose below 0.01 cc. In the mucosum, 0.01 cc. of the antiserum
failed to kill the organism.
In parallel series of experiments w i t h o u t the addition of complement, it was found t h a t the powerful immune sera (Nos. 4 and 8)
caused a considerable restraint upon the growth of the homologous
strains, the only difference between these sera and those containing
complement being t h a t their effects are less marked. When usedin quantities below 0.03 cc., neither of these sera caused a complete
suppression of the growth of organisms acted upon b y them. T h equestion naturally presents itself as to whether the suppression of
growth in cases where complement and the immune sera were employed was due to the actual destruction of the organisms or merely

I:IIDEYO NOGUCHI AND SEINAI AKATSU

781

to the agglutination. The latter assumption may be dismissed, as
the phenomenon, does not occur when the immune sera alone (containing the agglutinins) are used. Of course, the sparseness of colonies in these tubes must be partly due to the agglutination, whichtends to clump many thousand spirochetes to start a single colony.
Yet a total suppression cannot be explained on this ground, since asingle colony, once it has begun to grow, will finally form a diffuse
growth throughout the medium. As will be described later, the failure of the spirochetes to grow after being subjected to the action ofcomplement and immune serum was largely due to the actual destruction of the spirochetes. Syphilitic rabbit serum was also tested andshowed that there was an unmistakable destruction of the pallidum
strain in quantities above 0.03 cc. Normal rabbit serum showed aslight restraint upon growth of this strain when used in doses of 0.1
cc. Neither of these sera had any ill effect upon the spirochetes whencomplement was omitted from the mixture.

Microscopic Observations on the Effect of the Immune Sera upon
Spirochetes in Vitro.
Immune Sera 4 (Strain McD.), 8 (Strain XI), 10 (Strain B29),
and 12 (Strain Z. A.) were chosen for a series of observations inwhich we followed the changes which take place in Treponema pallidum when the organisms have been subjected to the action of thesesera. Three strains of the pallidum, McD., R, mad Z. A., were used
for the purpose. As in the previous experiments, the action of theimmune sera was studied with and without the addition of complement (guinea pig fresh serum).
The sera were inactivated at 56°C. for 30 minutes. The mixture of the serum,
spirochetal suspension (McD.), and complement (in cases where it was added)
was incubated in a water bath at 37°C. for 3 hours, and microscopic examinations
were made during and after incubation. Similar results were obtained with thestrains of R and Z. A.
Table VII demonstrates the fact that Treponema pallidum undergoes a fundamental change in its morphology when acted upon by
its immune sera and complement a t an optimum temperature. The
phenomenon is similar to the dissolution or disintegration of various ~782

IMMUNOLOGICAL STUDIES ON SPIROCHETESTABLE VII.
Effect of I m m u n e Sera upon Spirochetes i n Vitro.

Strain McD. 0.1 cc. + Immune Serum 4 (Strain McD.) 0.1 cc. +complement 0.1 cc.
Strain McD. 0.1 cc. + Immune Serum 8 (Strain XI) 0.1 cc. +complement 0.1 cc.
Strain McD. 0.1 cc. + Immune Serum I0 (Strain B29) 0.1 cc. +complement 0.1 cc.
Strain McD. 0.1 cc. + Immune Serum 12 (Strain Z. A.) 0.1 cc. +complement 0.1 cc.

The organisms werestrongly agglutinated within 1
hr., then they
became granularin appearance,and only a few
retained
their
form.

Strain McD. 0.1 cc. + Immune Serum 4 (Strain McD.) 0.1 cc. +no complement.
Strain McD. 0.1 cc. + Immune Serum 8 (Strain XI) 0.1 cc. +no complement.
Strain McD. 0.1 cc. + Immune Serum 10 (Strain B29) 0.1 cc. +no complement.
Strain McD. 0.1 cc. + Immune Serum 12 (Strain Z. A.) 0.1 cc. +no complement.

The agglutination
was even stronger
than in the above
series.
At theend of 24 hrs. theorganisms kept
their form.

Strain McD. 0 + no serum + complement.
"
"
0+ "
"
+ no "

No

noticeablechanges.

microorganisms under the influence of an immune serum. In theabsence of complement, the organisms were strongly agglutinated,but not dissolved, even after 24 hours.
In the following experiment, attempts were made to study thepart, if any, played by leukocytes in the destruction of the spirochetes
in vitro. Leukocytes both from normal and from immunized rabbits
were collected by Wright's method. The citrate blood was briefly
centrifuged, and the leukocytic layer of the sediment carefully skimmed and put into another centrifuge tube. In ordinary opsoninwork, the cells thus collected are used without washing, but in thepresent experiment, they were washed with a 0.9 per cent saline solution by a renewed centrifugalization. The immune leukocytes wereobtained from a rabbit which had been immunized with the Z. A.
strain (No. 12).
T h e technique was as follows: To 0.1 cc. of a suspension of one of the pallidum
strains were added 0.1 cc. of t h e leukocytic suspension, 0.1 cc. of each of t h e imm u n e sera, a n d 0.1 cc. of complement. T h e mixtures, after being well stirred,

783

HIDEYO NOGUCHI AND SEINAI AKATSU

were incubated in a water bath at 37°C. and examined at various intervals; forexample, 30 minutes, 1 hour, 2 hours, and 4 hours (kept at room temperature).
The examinations were made without staining under the dark-field microscope,and also as film preparations stained by Giemsa as well as by a modified Fontana
silver impregnation. Films prepared from a mixture in which unwashed citrateleukocytic suspension was used washed off the slides very readily and could not
be depended upon for accurate results. Normal as well as immune serum andnormal leukocytes were also used (Table V I I I ) .
TABLE

VIII.

Effect of Leukocytes upon Spirochetes in Vitro.
Strain McD.0.1 c c . + immune leukocytes (No. 12) 0.1 cc. + Immune Serum 4, 0.1 cc. + complement 0.1 cc.
Strain McD. 0.1 co. + immune leukocytes (No. 12) 0.1 cc. + Immune Serum 8, 0.1 cc. + complement 0.1 cc.
Strain McD. 0.1 cc. + immune leukocytes (No. 12) 0.1 cc. + Immune Serum 10, 0.1 cc. + complement 0.1 cc.
Strain McD.0.1 c c . + immune leukocytes (No. 12) 0.1 cc. + Immune Serum 12, 0.1 cc. + complement 0.1 cc.

Strong agglutination and graduallysis, many spirochetes
adhering to the leukocytes, into whichsome were deftnitely ingested.
Total disappearance of spirochetes in 24 hrs.

Strain McD. 0.1 cc. + normal leukocytes 0.1 cc. +Serum 4, 0.1 cc. + complement 0.1 cc.
Strain McD. 0.1 cc. + normal leukocytes 0.1 cc. +Serum 8, 0.1 cc. + complement 0.1 cc.
Strain McD. 0.1 cc. + normal leukocytes 0.1 cc. +Serum 10, 0.1 cc. + complement 0.1 cc.
Strain McD. 0.1 cc. + normal leukocytes 0.1 cc. +Serum 12, 0.1 cc. + complement 0.1 cc.

Strong agglutination and generaldisintegration of
spirochetes but
decidedly
lessthan in the preceding
series.
Phagocytosis
present.

ImmuneImmuneImmuneImmuneStrain McD. 0.1 cc. + no leukocytes + Immune Serum 4, 0.1
cc. + complement 0.1 cc.

Much lysis in 24
hrs.

Strain McD. 0.1 cc. + immune leukocytes 0.1 cc. + no immune
serum + complement 0.1 cc.

N o striking
changes.

Strain McD. 0.1 cc. + immune leukocytes 0.1 cc. + no immune
serum + no complement.

No lysis or phagocytosis.

Strain McD. 0.1 cc. + immune leukocytes 0.1 cc. + normal
serum 0. l c c . + complement 0.1 cc.

No lysis or phagocytosis.

Strain McD. 0.1 cc. + immune leukocytes 0.1 cc. + normal
serum 0.1 cc. + no complement,

No lysis or phagocytosis.

784

IMMUNOLOGICAL STUDIES ON SPIROCHETESParallel series of experiments were also carried out with Strains
R and Z. A. with similar results. It seems apparent, therefore, thatsome of the spirochetes are readily taken up by immune as well as
normal rabbit leukocytes, when an immune serum and complement
are simultaneously added to the mixture. The number of spirochetes
taken up by phagocytes is small compared with what we are accustomed to see with bacteria. It may be due to the filamentous feature of the organisms, which prevents the cells engorging them withreadiness, or what appears still more probable is that they form
enormous masses of entangled nets too large to be taken up by thephagocytes, and then gradual lysis occurs. It is also possible thatthe engorged spirochetes quickly disappear through intracellulardigestion. At all events, the agglutinated masses become gradually
granular and indistinct and finally undergo dissolution. The completeness with which this process proceeds is most marked when
immune leukocytes are used. Perhaps there exists within thesecells certain elements which render the lytic processes more energetic
than when only immune serum and complement are used. In theabsence of complement, the immune serum produced only a slightdegree of phagocytosis and no lysis. The organisms, after being
acted upon by the leukocytes in the immune serum and complement
mixture, lose their property to take Giemsa stain as strongly as those
not so treated. Their curves are seen to be flattened in many specimens. They still retain their affinity for the silver precipitation
method (Fontana). It has been difficult to stain the spirochetes
distinctly within the phagocytes, although they could be seen in fresh
preparations by means of dark-field illumination. The large number of spirochetes adhering to the leukocytes prevents a clear appearance of the phenomenon.
A series of experiments was performed with normal human leukocytes in conjunction with the immune rabbit sera and complement.
The results were similar to those recorded for normal rabbit
leukocytes.

HIDEY0 NOGUCHI AND

SEINAI A K A T S U

785

Effects of Immune Sera and Leukocytes upon the Uncultivated Strainof Treponema pallldum.
T h e action of the sera derived from rabbits immunized with cult i v a t e d strains of Treponema pallidum m a y also be studied. I t m a y
be tested b y subjecting a suspension of a virulent testicular material
rich in the pallidum to the effect of the i m m u n e sera and theninoculating certain animals susceptible to experimental syphilis.
Another procedure would be to s t u d y the i m m u n i t y phenomena which
follow the mixing of the pallidum and the serum in vitro. Agglutination, immobilization, granular disintegration, dissolution, or complement binding m a y be studied. T h e first m e t h o d is subject to thedifficulties inherent in experiments in which the susceptibility of different individuals constitutes an inconstant factor. I n fact, ourpreliminary experiments indicated t h a t s t u d y with extensive m a t e rial is required. This phase of the s t u d y has been u n d e r investigation b y one of us since 1911 and will have to be continued for a longer
time. T h e test-tube p h e n o m e n a were b e t t e r observed.
To 0.1 cc. of a rich suspension of virulent pallidumfrom syphilitic orchitis of a
rabbit, 0.1 cc. of each of the several immune sera, comprising Nos. 4 (Strain McD.),
8 (Strain XI), 12 (Strain Z. A.), and 10 (Strain B29) was added, and 0.1 cc. of a 40
per cent solution of fresh guinea pig serum as complement. The mixture was madeup to 1 cc. by adding a 0.9 per cent sterile saline solution and then placed in awater bath kept at the temperature of 37°C. The mixture, an opalescent fluid,
was examined from time to time for the agglutination, motility, or disintegration of the pallidumunder the dark-field microscope. Controls with the pallidum
alone, the pallidum with the immune serum (inactivated), and the pallidum
with the complement were provided.
The results of our observations were in the main similar to those obtained byZinsser, Hopkins, and McBurney, except for a few points. It was noticed inour experiments that the particular strain of Treponemapallidum employed became sooner or later immobilized by the addition of the immune sera, and none
was motile after 3 hours at 37°C. In the control tubes containing plain saline orcomplement solution, the organisms were still active for several hours longer. In
some control tubes, there were a few motile pallida at the end of 24 hours. The
immobilizing effect was augmented by the presence of complement. There wasno definite agglutination or dissolution of the pallidum except in cases of
Sera 4 and 8, where there were small clumps of entangled immobilized organisms
suggesting a slight agglutination. But we were unable to find definite disintegration of the pallidum.

786

IMMUNOLOGICAL STUDIES ON SPIROCHETESStudies were also made to discover whether or not leukocytes
from an immune or normal rabbit ingested the tissue pallidum under
the influence of the immune serum, but the results were inconstant.
Although there was an unmistakable phagocytosis in the presenceof the immune serum, it was slight in comparison with the cultivatedstrains, and there was no general disintegration of the organisms.
In considering the above results, it appears as though the immune
sera prepared by injecting the rabbit with avirulent strains of culture
pallidum exerted much less effect upon a virulent strain derived froma syphiloma in a rabbit. Whether or not this is due to unsuitable
immunization or to a modification in strains is not shown by the present experiments. The failure of agglutination of the tissue pallidum
m a y have been due to the presence of various tissue proteins simultaneously introduced into the mixture, for these apparently indifferent substances can often interfere with agglutination or complement fixationY The entire question as to the relation between theuncultivated virulent strains and those which had become avirulent
through cultivation is still under investigation.
CONCLUSIONS.

Experiments were carried out for the study of culture spirochetes
in their relation to various immunity reactions in vitro. Several
strains of Treponema pallidum and one each of Treponema calligyrum,
Spirochceta refringens, Treponema microdentium, and Treponema
mucosum were used. Tests were made of immune substances responsible for agglutination, complement fixation, spirocheticidosis,
and opsonization. In cases of agglutination and complement fixation, cross titrations were made.
1. In the sera derived from rabbits immunized with various spirochetes agglutinins were demonstrated in varying quantities for thehomologous antigens. The amounts of agglutinins developed wereconsiderably higher in the pallidum immune sera than in the othergroups. There was no parallelism between the amounts of antigens
injected and the amounts of agglutinins developed.
2. Cross titrations among different pallidum strains revealed that
23 Noguchi, H., and Bronfenbrenner, J., J. Exp. Med., 1911, xiii, 92.

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the agglutination is not necessarily strongest when homologous
antigens and immune sera are brought together.
3. On the other hand, the reactions between the immune sera
and antigens belonging to different species were sufficiently specificto justify the grouping.
4. Certain degrees of group reaction were observed between thepallidum immune sera and the calligyrum, and occasionally very
faintly also between the pallidum and the refringens antigens andvice versa. There was a much more pronounced group reaction between the calligyrum and refringens. The immune serum and antigen of the microdentium showed a slight affinity for the mucosum but
none for the patlidum, calligyrum, or refringens, while the mucosum
immune serum caused a slight agglutination with many membersof the other groups. Hence, it appears that the pallidum is more orless related to the caItigyrum, while the affinity between the calligyrum and refringens, and possibly also between the calligyrum andmucosum in a much smaller degree, seems close. The microdentium
showed the least relation to any other spirochetes.
5. Titration of agglutinins in the sera obtained 3 months after the
cessation of immunization revealed that the agglutinin contents werealready greatly reduced, having fallen roughly to 0.01 of the original
strength. The rates of disappearance were irregular in different
animals and bore no direct relation to the initial fiters. Titration
made of the immune sera which had been preserved aseptically in arefrigerator (6°C.) during the same period (3 months) indicatedthat the original strength of these serawas reduced to about one-tenth.
The agglutinins for spirochetes disappear from the rabbit's body much
more rapidly than they are reduced in the separated sera by deterioration on standing at 6°C.
6. Titration of the immune sera for complement fixation power
showed with a few exceptions, in which there was only slight complement binding, that the titers were high enough to indicate the presence of this principle. The anti-paltidum sera possessed higher average titers than the other immune sera tested with correspondingly
homologous antigens. The least active were the anti-refringens sera.
7. Cross titration of anti-palliclum immune sera for complement
fixation showed that a given sermn with a high titer for its own
strain of antigen was also strong with most of the other strains of the788

IMMUNOLOGICAL STUDIES ON SPIROCHETES

pallidum. Instances occurred also in which the titers with heterologous pallidum antigens fell far below those of the homologous. Groupreactions between the different spirochetes, such as the pallidum andthe calligyrum, the calligyrum and the refringens, and the microdentium and the mucosum, were also indicated. The mucosum and thepallidum showed a slight degree of group reaction. No anti-pallidum
serum fixed complement with the microdentium.
8. The immune sera were tested for their spirocheticidal properties

in vitro against the correspondingly specific and heterologous varieties with and without the addition of complement. Many of the antipallidum sera killed their own strains. Normal rabbit serum exhibited only a slight degree of inhibition. Without complement,
the immune sera caused a considerable reduction in the number ordensity of colonies, but not a complete suppression of growth. Complement alone had no injurious effect upon the pallidum strains.
The antisera for the calligyrum, refringens, and mucosum showedfeeble spirocheticidal action, while the antisera for microdentium
was stronger. A syphilitic rabbit serum tested against a strain of
culture pallidum gave a feeble inhibitory effect.
9. Under the influence of immune sera and complement, the spirochetes undergo within a few hours complete disintegration or granulardegeneration. Without complement, they are more powerfully
agglutinated, but no disintegration occurs, even after 20 hours, andcomplement alone has no effect.
10. In the presence of homologous immune serum and complement, the culture pallidum may be ingested by the leukocytes, but
phagocytosis is slight, possibly on account of the filamentous natureof the organisms. The spirochetes in such a mixture disintegrate
within a few hours, disintegration being especially rapid when the
immune leukocytes are used. In the absence of immune sermn,
phagocytosis is not noticeable, while without complement but in thepresence of immune serum and leukocytes, some phagocytosis,
without subsequent lysis, occurs.
A virulent strain of pallidum, obtained from syphilitic orchitis ina rabbit, exposed to agglutination, lysis, and phagocytosis by animmune serum prepared by means of culture pallidum strains, showed
only slight agglutination and phagocytosis but rapid immobilization
without disintegration in the presence of complement.