野口英世著

Journal of Experimental Medicine

所収論文

こ の PDF は Journal of Experimental Medicine に 掲 載 さ れ た 論 文 を Rockefeller
University Press の許可（2020 年 3 月 18 日付）を得てアップロードしています。

ETIOLOGY OF YELLOW FEVER.
XII.

CHEMOrm~A.PY VERSUS SEROTHERAPY IN EXPERrM~.NTAL
INFECTION WITH LEPTOSPIRA ICTEROIDES.

BY HIDEYO NOGUCHI, M.D.
(From tie Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, April 3, 1920.)

The work of Ehrlich and Hata t on the therapeutic effects of arsenicalorganic compounds in various infections caused by spirochetal or protozoan organisms has led to considerable investigation by later workers, notably Jacobs, Heidelberger, Amoss, and Bull~-5 and Pearce andBrown, ~ of the extent to which this kind of curative method is applicable. The present experiments were undertaken, not as research inchemotherapy, but merely as part of a routine study of experimental
infection in guinea pigs with Leptospira icteroides, derived from certain
cases of yellow feverY It was of particular interest to ascertain how
Leptospira icteroides would behave toward the two widely employedchemotherapeutic agents, salvarsan and neosalvarsan, and what
difference, if any, there is between its behavior and that of the inciting
agent of infectious jaundice in this respect, for the latter has been
extensively studied by Inada, Ido, and their coUaborators, 8 as well ast Ehrlich, P., and Hata, S., Die experimenteUe Chemotherapie d~r Spirillosen,
Berlin, 1910.
Jacobs, W. A., J. Exp. Med., 1916, xxiii, 563.
8 Jacobs, W. A., Heidelberger, M., and Amoss, H. L., J. Exp. Med., 1916,~ i l , 569.
4 Jacobs, W. A., Heidelberger, M., and Bull, C. G., J. Exp. Med., 1916, ~iil,577.
5 Jacobs, W. A., and Heidelberger, M., J. Biol. Chem., 1915, xx, 513, 659, 685;
xxi, 103, 145, 403, 439, 455, 465.
e Pearce, L., and Brown, W. H., J. Exp. fried., 1918, xxviii, 109.
7 Noguchi, H., J. Exp. Med., 1919, xxix, 547, 565, 585; xxx, 1, 9, 13, 87, 95,
401; 1920, xxxi, 135, 159.
s Inada, R., Ido, Y., Hold, R., Kaneko, R., and Ito, H., J. Exp. Med,, 1916,
x~i, 377. Kaneko, R., and Okuda, K., Verhandl.japan. park. Ges., 1916, vi, 49.
381

382

ETIOLOGY Ol~ YELLOW FEVER. XlI

by European investigators, especially Uhlenhuth and Fromme) A
large number of experiments with salvarsan and neosalvarsan, atoxyl,and arsacetin against Leptospira icteroh¢emorrhagi~ was also carriedout in 1917, with negative results. 1° Being regarded by some as a
spirochete, Leptospira icteroh~emorrhagi~z was expected to yield tochemotherapy by means of salvarsan or its derivative, but Inada
and Ido and others soon found that neither salvarsan nor neosalvarsan
had any definite therapeutic value in infections with this organism.
In this respect, at least, the organism discovered by Inada and Ido
in infectious jaundice did not behave like the other pathogen!c spirochetes, and this characteristic may be regarded as giving further support to the opinion that the leptospira group of organisms forms atype of its own and differs from the other pathogenic genera of Spirochmtoidea. The experiments here reported concern the behavior of
Leptospira ict~roides toward the arsenical compounds, not only inthe animal body, but also in ~itro, and the effect upon the organism of
salvarsanized serum. From the practical standpoint it seemed advisable to include also a brief comparison of the action of the arsenicalpreparations and that of anti-icteroides immune serum upon Leptosplra
icteroides in experimental infection and in vitro.

Effect of Salvarsan and Neosalvarsan upon the Course of Experimental
Infection oJ Guinea Pigs Inoculated with Leptospira icteroides.
Several series of experiments were performed in order to ascertain
whether administration of salvarsan or neosalvaxsan simultaneouslywith the inoculation or shortly afterwards would in any way influence
the development of the experimental leptospiral infection in guinea
pigs. If the virulence of Leptospira icteroides for this animal wereconstant a few series of experiments only would have been sufficient
to determine the point, but owing to the variable character of thepathogenicity of the organism for individual guinea pigs it was necessary to repeat similar experiments. In some series control animals
survived or escaped infection, hence the interpretation of the effectof the drugs was rendered inconclusive. In the earlier experiments
9 Uhlenhuth and Fromme, Z. Immuni~tsforsch., Orig., 1916, xxv, 418.
x0Noguchi, H., unpublishedresults.

383

HIDEYO NOGUCHI

the amounts of infecting material were near a single lethal dose, evensubminimum lethal doses being used; i.e., a q u a n t i t y capable ofproducing in the m a j o r i t y of instances a mild infection with recovery.
T h e mode of experiment was practically the same i n all the series.
I n one instance an unneutralized solution of salvarsan was employed,
othervdse salvarsan has been used as an alkaline solution. T h e
injection of the infecting material was intraperitoneal and t h a t ofthe drugs subcutaneous.
TABLE I.
Inocula~on of Injecting Material into Guinea Pigs Followed by Injection of Drug.

November 27, 1919. The amount of infecting material used was 0.5 ce.
Amount ofsresminolnatrium.

Course of disease.

Result.

Remarks.

gm.

0.03

Mild; 6 to 8 days.

0.02
0.01

Moderate (Jr); 6 to 11 days.
Severe (Jr Jrjr Jr).; 6 to 11 days.

0.005
0.0025

Mild; 7 to 9 days.
Severe ( + + + + ) ; 6 to 11 days.

0.001
Died in 6 days.
0.0005" " 9 "
Control. Severe (jr Jr Jr); killed in 6 days.
Moderate (jr Jr); 7 to 8 days.
C~
Mild (~-); 7 days.

Recovered. Died in 15 days; no jaundice.
No jaundice.
Died in 13 days; jaundiCe
fading.
No jaundice.
Killed in 11 days; typicallesions.
TypicaL
Recovered.

Series/.--November 27, 1919. The infecting material consisted of a mixtureof citrate blood (showing the leptospiras) of a guinea pig experimentally infectedwith Guayaquil Strain I and a rich culture of the same strain. Ten guinea pigs
were inoculated intraperitoneally, each with 0.5 cc. of the mixture, and all but
three.(controls) then received subcutaneously a solution of arsamlnolnatriun~ (aJapanese preparation of neosalvarsan), the quantities given ranging from 0.03
to 0.0005 gin. (Table I).

T h e a m o u n t of infecting material used in this series was near asingle m i n i m u m lethal dose, as shown in the control animals and also
in those which received the smallest amounts of the drug. I t is
n o t e w o r t h y t h a t in the three guinea pigs which received 0.03, 0.02,

384

ETIOLOGY

OF Y E L L O W

FEVER.

XlI

and 0.005 gm. of the drug, respectively, there was no jaundice at a n yperiod of the infection.

Series 2.--December 3, 1919. The infecting material consisted of a mixtureof 1 cc. each of cultures of Guayaquil Strains 1 and 5, and 7 cc. of heart's bloodfrom a guinea pig infected typically with Strain 1. 0.5 cc. of the mixture was
inoculated intraperitoneally into each of sixteen guinea pigs. One group of eightanimals was then inoculated subcutaneously with an unneutralized solution ofsalvarsan and the other group of eight with the solution of neosalvarsan in doses
TABLE II.

Inoculation of Infecting Material into Guinea Pigs Followed by Injection of Drug.
December 3, 1919. The amount of infecting material used for the treatedanimals was 0.5 ec.
Treated animals.

Amountof
drug.

Untreated controls.

Course of disease.
Salvarsan(acid solution).

Neosalvarsan.

Test forvirulenceof
infectingmaterial
used....._L_.

Course of disease.

cc.

0.03

Mild; recovery.

Mild; recovery.

1.0

0.02

No symptoms.

Severe ( + + + ) ; recov-

0.5
0.2

Severe ( + + + + ) ; recovery.
Mild; recovery.
Moderate ( + + ) ; recov-

0.1

No symptoms.

ely.

0.01
0.005
0.002
0.001
0.00050.0002ca*~

Mild; recovery.
No symptoms.

ely.

Died in 12 days;
typical.
No symptoms.
Died in 13 days; typical.
Severe ( + + + ) ; recovery.

varying from 0.03 to 0.0002 gm. There were four control guinea pigs which received varying doses of the infecting material and were not treated with the drug
(Table II).
T h e results of these experiments suggest a possible slight protective
effect of the drugs in some individuals, although, from the mildnessof the infection in the control animals the survival of those individuals
m a y also be explained on the basis of a natural resistance to Lepto-

HIDEYO NOGUCHI

385

spira icteroides.

I t is of interest to note t h a t m o r e guinea pigs a m o n g
those t r e a t e d with salvarsan escaped the infection t h a n a m o n g thoset r e a t e d with neosalvarsan. T h e indecisive character of the experim e n t s m a d e necessary a n o t h e r series with multiple m i n i m u m lethal
doses. I n Series 3 at least 50 m i n i m u m lethal doses were used foreach guinea pig.

Series 3.--March 13, 1920. The infecting material consisted of a mixture of
5 cc. of a culture of Guayaquil Strain 1, 5 cc. of kidney emulsion, and 5 cc. of
citrated heart's blood from a guinea pig inoculated with the same strain 4 days
previously. In order to produce a fatal infection in guinea pigs 0.5 ce. of the
mixture was inoculated intraperitoneaUy into each animal. As the protocol
shows, 0.01 cc. of this material killed a control animal in 7 days with typicalsymptoms, and a much smaller quantity would have been sufficient to cause a fatal
infection, although the minimum lethal dose was not determined in the present
series of experiments.
Salvarsan was dissolved in sterile distilled water and a 0.1 N sodium hydroxide
solution was gradually added until the precipitate first formed was completely
redissolved. The final concentration of the drug was made 0.1 gin. per 10 co.
(stock solution). Neosalvarsan was dissolved in sterile distilled water in the ratioof 0.1 gin. per 10 cc. (stock solution). Further dilutions were made with 0.9
per cent salt solution.
The animals were inoculated with 0.5 cc. of the infecting material intraperitoneally, and within about 30 minutes varying amounts of salvarsan and neosalvarsan solution were injected subcutaneously. The amounts of the drug were0.00005, 0.0001, 0.0002, 0.0005, 0.001, 0.002, and 0.003 gin. for guinea pigs of
350 gin., and each dose was contained in from 0.5 to 3 cc. of fluid, according toconvenience in measurement. Table I I I gives the results.
Series 4.--March 18, 1920. Although it was still too early to know the resultsof the series of March 13, another series was begun on March 18 in which larger
doses (0.01, 0.02, and 0.03 gln.) of salvarsan and neosalvarsan were used. The
solutions of the drugs were freshly prepared from another set of ampules, andthe infecting material consisted of a guinea pig kidney emulsion of Guayaquil
Strain 1 of Leptospira icteroides. Other technical details were the same as in
the previous experiments. Table IV gives the results.
T h e experiments of Series 3 (March 13) show t h a t guinea pigs
receiving a t least 50 m i n i m u m lethal doses of a strain of Leptosp4ra
icteroides intraperitoneally and v a r y i n g quantities of salvarsan orneosalvarsan subcutaneously within 30 m i n u t e s f r o m the t i m eof inoculation of icteroides undergo a typical course of leptospira
infection, resulting in the m a j o r i t y of instances in d e a t h within a386

ETIOLOGY

OF Y E L L O W

FEVER.

Xll

period the variations of which m a y be considered usual in such aseries of experiments. W i t h salvarsan there were two instances ofrecovery after a severe infection coinciding with elevated doses ofthe drug (0.001 and 0.003 gin.), while in animals receiving less of the
TABLE IIl.

Inoculation of Infecting Material into Guinea Pigs Followed by Injection of Drug.
March 13, 1920. The amount of infecting material used for the treatedanimals was 0.5 ce.
U n t r e a t e d controls.

T r e a t e d animals.

Course of disease.
Amountofdrug

Course of disease.

Salvarsan.

Ncosalvarsan.

0.00005 Died in 5 days; typical. Recovery after a mild
infection, with definite jaundice from
Mar. 20-24, 1920.
a
tt 8
tt
.
Died in 5 days; typical.
0.00010.0002

~c

~t 5

cc

Died in 6 days; typical.
Died in 7 days; typical.
Died in 7 days; typical.

~c

O.0OO50.001 Recovery after a typical infection, with intense jaundice from
Mar. 19-25, 1920.
Died in 6 days; typical. Recovery after a mild
0.002
infection, with jaundice from Mar. 1923, 1920.
0.003 Recovery after a typical Recovery after a severe
infection, with ininfection lasting fromtense jaundice from
Mar. 18-20, 1920.
Mar. 18-23, I920.
drug d e a t h occurred in two instances 2 d a y s earlier t h a n in controls,a fact w o r t h y of notice. W i t h neosalvarsan the n u m b e r of recovered
animals was three, two with the largest doses of the drug (0.002 and0.003 gm.) a n d one with the m i n u t e s t dose employed (0.00005 gin.).

HIDEYO NOOUCHI

387

All the rest died in from 5 to 9 days with typical symptoms. The
results both with salvarsan and with neosalvarsan suggest that thesearsenical compounds, when employed in certain quantifies, may
somewhat modify the severity of the infection and occasionally savea guinea pig from death, although failing to suppress the infection
completely.
The results of the experiments of Series 4 (March 18) were lessfavorable than those of Series 3. Here, of the two controls one diedin 12 days and the other recovered after a definite infection. Two
TABLE IV.

Inoculation of Infecting Material into Guinea Pigs Followed by Injection of Drug.
March 18, 1920.
animals was 0.5 cc.

The a m o u n t of infecting material used for the treatedTreated a~imal~.

Untreatedcontrols.

Course of disease.
Course of disease.

"$alvarsan.

Neosalvarsan.
G&

8m°

0.01

Died in 11 days; typical. Died in 12 days; typical.

0.~

0.02

Had fever but no jaun- " " 12 ""dice; recovery.
Died in 11 days; typical. Had fever but no jaundice; recovery.

0.~

0.03

Died in 12 days; typical.
Recovery after a deftnite infection, withintensejaundicefrom Mar. 3 0 - 3 1 ,1920.

of the three guinea pigs treated, either with salvarsan or neosalvarsan,died in from 11 to 12 days, and the one animal which survived in each
set had fever indicative of an abortive leptospira infection.
From the therapeutic standpoint neither salvarsan nor neosalvarsan
is of any value in experimental infections of guinea pigs with Leptostzira i~teroides. In contrast, it may be interesting to include herea protocol illustrating the highly specific protective value of an anti~teroides serum (horse). The amount of the immune serum required
to protect a guinea pig from an infection with Leptospira icteroides
is exceedingly minute.

388

ETIOLOGY OF YELLOW I~'EVER. XlIContrasted Effect of Anti-icteroides Immune Serum in Vivo.
March 11, 1920. The experiments with the immune serum were undertakenwith the same strain of L e p t o s p # a icteroides (Guayaquil No. 1) that was usedin the chemotherapeutic experiments already described. The material was a
mixture of the emulsions of kidney and liver from a guinea pig which was sho~cingearly symptoms of the leptospira infection. As the protocol shows, the minimum
lethal dose of this emulsion was such that 1 cc. of a 1 : 10,000 dilution of it killed
a guinea pig with typical symptoms in 12 days, and the same quantity of a 1 : 1,000
or 1:100 in 10 days. 0.5 cc. of the original emulsion killed a guinea pig in 8 days.
TABLE V.
Inoculation of Infecting Material into Guinea Pigs Followed by Injection of A n t i icteroides Serum.

March 11, 1920. The amount of infecting material used for the treatedanimals was 0.5 cc.
Treated animals.

Antl-lcteroSdes immune

serum.
Dilution.
Amount.

Course of disease.

Untreated controls.
Test forvirulence of
infectingmaterial used.
cc.

CO,

1

Course of disease.

1:100,000 Recovery after a mild infection.
1 : 10,000
No symptoms.
""(suspicion
1:1,000of trace of jaundice on
Mar. 20, which had disappeared the following
morning).
1:100
No symptoms.

0.5
0.1

0.01
0.001
0.00010.00001
0.000001

Died in 8 days; typical.
Recovery after a severe infection (exceptional resistance).
Died in 10 days; typica!
" " 10 """ " 12 ""No symptoms.

1:10

For infecting guinea pigs to be treated with anti-icteroides serum, 0.5 cc.
(about 5,000 minimum lethal doses) of the same emulsion was intraperitoneallyinjected. The different amounts of the anti-icteroides serum (collected fromHorse 2 on February 25, 1920) were then injected, also intraperitoneally, within
half an hour after the inoculation. The amounts of immune serum were 0.00001,
0.0001, 0.001, 0.01, and 0.1 cc. Table V is a record of the results.

According to a conservative estimate, therefore, the power of theimmune serum is such that 1 cc. of a 1:10,000 dilution prevented an
infection when the dose of the infecting material was 5,000 minimum

~mr.Yo ~oG~cm

389

lethal doses. In other words, 1 cc. of the serum had the power toprotect a guinea pig of 350 gm. body weight against 50,000,000(10,000 X 5,000) minimum lethal doses when administered intraperitoneally 30 minutes after intraperitoneal inoculation. The
specific protective property of the immune serum is indisputably
highly efficacious as compared with salvarsan or neosalvarsan, thevalue of which is at least doubtful. A point of considerable importance is that in certain guinea pigs receiving small quantities of salvarsan and neosalvarsan the period before death seemed to be shortened by 2 days (5 days), as compared with the average period in
untreated control animals (7 days). It may be that the predilectioa
of arsenic compounds for the renal tissues had a definite predisposing
effect, due to chemical injury, upon this organ, which Leptospirc~icteroides also attacks particularly.

Direct A ction of Salvarsan and Neosalvarsan upon Leptospira icteroides.
Ehrlich makes a special point of having found, in his quest forchemotherapeutic agents, that a preparation whose destructive power
on a microbic organism is great in vitro may have no, or only a slightantagonistic effect when introduced into the animal body, or the
relation between the effect manifested in vitro and in vivo may be thereverse. Ehrlich's efforts to find a chemotherapeutic preparation
were principally directed toward its effect in vivo, since the pathogenicparasites with which he was working belonged to the class of protozoa
or a class closely allied to it, and there were no virulent cultures on
hand to be tested in vitro as well as in vivo. Certain arsenic compounds elaborated by Ehrlich and his coworkers displayed a highly
sterilizing effect on various spirochetoid organisms when introduced
into the animal body infected with them, while the direct effect ofthese preparations upon the same organisms in vitro was almost nil.
Ehrlich interpreted the highly parasiticidal properties of these compounds as due to a certain modification (reduction) in the animal
body of substanCes otherwise comparatively inert. Salvarsan andTreponema pallidum constitute a good example in point.
Leptospira icteroides is resistant to saponin, n a property which alone
would serve to differentiate it from certain other groups of spiro.
11Noguchi,H., J. Exp. Med., 1919, xxx, 13.

390

ETIOLOGY 01' YELLOW FEVER.

Xllchetoid organisms (Treponema and S p l r o n e m a ) . T h e fact t h a t i nvivo the leptospiras were scarcely influenced b y the introduction ofsalvarsan or neosalvarsan constituted another point of dissimilarity
between the leptospira and other spirochetal parasites. Hence theeffect of salvarsan or neosalvarsan upon the leptospira from yellow
fever cases in vitro presented a further interesting subject for study.
Several experiments were also performed to determine the b e h a v i o rof blood serum derived from a rabbit which had been injected intravenously 1 hour previously with salvarsan or neosalvarsan in considerable quantities; t h a t is, a s t u d y was m a d e of the effect of salvarsanized serum, as well as of drug solutions, upon icteroides i n vitro.
TABLE VI.
Addition of Drug to Culture of Leptospira icteroides.

November 26, 1919.
Final concentration ofarsaminolnatrlum.*

Result.

1 : 2,000
1: 6,000

All inactive and most have disappeared.
""" somedegenerated.

1: 20,000
1 :60,000

1:200,000
1: 600,000
1: 2,000,000
Water (control).

"

"

¢¢

¢¢

thinner, and smoother.

For the most part inactive.
~,

~¢

~c

¢c

"

"

"

" active, a few inactive.

cc

some

still

active.

* The concentration resulting from the mixture of the drug solutions with theculture is given.
To each of several tubes containing 1 cc. of a culture of Guayaquil Strain 6
w a s added 1 cc. of different dilutions of salvarsan, neosalvarsan, or arsaminol-

natrium. The mixtures were allowed to stand at room temperature and examined
after 2 or 2½ hours, and again after 24 or 48 hours. As Tables VI and VII show,
sufficient quantities of the drugs rendered the leptospiras immotile, and brought
about general disintegration. The highest dilution of any of the drugs whichstill killed the organism was about 1:200,000.
I n testing the leptospiriddal strength of the solutions of salvarsano r neosalvarsan, it was i m p o r t a n t to take note of the reactions of thesolutions. Salvarsan, according to the usual practice, is first t r e a t e dwith sodium hydroxide solution until completely precipitated out

EmEYONOOUC~

391

a t the point of neutral reaction. In order to obtain a clear solutioni t is necessary to add more alkali until the precipitate completely
dissolves. At this point the solution is no longer slightly, but intensely alkaline. By reducing the alkalinity with hydrochloric acidto a reaction of about pH 8 a bulky precipitate is once more formed,
and at pH 7 the entire substance flocculates out of the solution. In
making a microbicidal titration a clear alkaline solution was used inascending dilutions to eliminate the destructive effect of the reaction
alone. It was found that the 1:1,000 dilution was still too strongly
TABLE VII.

Addition of Drugs to Cul~re of Leptospira iaeroides.
December 3, 1919.
Final
concentrationof the drug.

1":200

Salvarsan.
2] hrs.

Neosa]varsan.
24 hrs.

2~ hrs.

24 hrs.

Precipitate (?).

Precipitate
All inactive.
All immobilized.
heavy.
~cc~
1: 2,000
Some
active; Allimmobile;
precipitate.
precipitate.
immobile;
1:20,000
Some
active; All
precipitate.
precipitate.
¢c¢#
1: 200,000
For the most All immobile.
part active.
1: 2,000,000 For the most For the most For the most Few active.
part
immopart active.
part active.
bile; few acfive.
Saline (con- All active.
All active.
All active.
For the most parttrol).
active.

alkaline to be used (far beyond pH 8), the leptospiras dying rapidly
in such a solution, while dilutions higher than 1:10,000 had a reaction
which was practically that of the diluent (saline solution), pH 7.
Neosalvarsan dissolved in distilled water in 1:100 dilution is a clear
amber-yellow and shows to phenol red an intense red color; in diluting
to 1:1,000 with saline solution its reaction approaches pH 7.2; in
1:10,000 dilution it is no longer perceptible to phenol red. In actual
experiments, however, 1 cc. of each of the dilutions of the drugs was
mixed with 1 cc. of a rich culture of one of the ~t~ro/~es strains which

392

ETIOLOGY OF YELLOW FEVER. XlI

had a reaction of pH 7.4. This mixing brought down the pH valueof the lower dilutions and raised t h a t of the higher. The reactions
between p H 7 and p H 7.8 are well borne by icteroides, the optimum
being near p H 7.2 to 7.4. A reaction beyond p H 8 or below p H 6.6
is unsuitable for the existence of the organism. The devitalizing
action of salvarsan and neosalvarsan, even in optimum reactions,is rather slow, a contact of m a n y hours being required before deathensues, as the protocol shows.
TABLE VIII.
Addition of Drugs to Culture of Leptospira icteroides.
March 18, 1920.
Final

Salvarsan.

Neosalvarsan.

c o n c e n t r a t i o n of

the drug.

1:200

Reaction.

Intensely

A f t e r 15 min. A f t e r 18 hrs.

Dead.

SlightlypH
over8.

alka°

1: 2,000

Reaction. After15 mln. A_f t e r_18 h r s .

line.
Intensely

Dead.

Acl,lve"

pH 7.2alkaline.

1: 20,000
pH 7.2
1: 200,000
pH 7.2
1: 2,000,000 pH 7.2
Saline control pH 7.2(no drug).

Active.

Dead.

g4

~4

pH 7.2pH 7.2. . . .
(?). pH 7.2,4
Active. pH 7.2

4~

4¢

4~

"

g~

~C
J

Active.*44* All found dead after 96 hours; the control tube was lost through contamination.
To each of several tubes containing 1 cc. of the same culture of icteroides,
Guayaquil Strain 5 (pH 7.4), was added 1 cc. of several different dilutions of drugs,
1:100, 1 : 1,000, 1:10,000, 1:100,000, and 1:1,000,000 of alkalinized salvarsan solution and neosalvarsan. The mixtures were allowed to stand at 26°C. and thecontents examined under the dark-field microscope after 15 minutes and again.
after 18 hours. The results are recorded in Table VIII.
I t is evident t h a t Leptospira icteroides is highly sensitive to theaction of salvarsan and neosalvarsan, but their action is comparatively
slow, requiring m a n y hours contact. The effects of the drugs in a
culture medium are found to be approximately the same as in thecase of direct mixing of culture and solutions.

HIDEYO NOGIYCHI

393

Various dilutions of the drugs were added to the usual medium (rabbit serum,
25 per cent, agar, 0.3 per cent, total volume, 6 cc.). The culture used was Guayaquil Strain 1, and the tubes were allowed to stand for 5 days at room temperature(26°C.). A medium containing salvarsan or neosalvarsan in a ratio of more than
1:200,000 was found to be unsuitable for the growth of the organism (Table
IX). No attempt was made to determine quantitatively the exact leptospiricidal
titers of the two drugs.
TABLE IX.

Addition of Drugs to Culture Media.*
March 6, 1920.
Final

concentrationofthe drug.

1:600
1:1,800
1: 6,000
1:18,000
1:60,000
1:180,000
1: 600,000
1:1,800,000
1: 6,000,000
Control.

Salvarsan.
BmNeosalvarsan.
m
m
m

m
m

m
N

+
+
+
+
+

<+
+
+
+
+

* The medium consisted of rabbit serum, 25 per cent, agar, 0.3 per cent, total
volume, 6 cc.
The changes in the color of salvarsan, when observed after 12 days standing,were noticeable in the tubes containing dilutions from 1:100,000 up, while withneosalvarsan the changes were not found in dilutions below 1:10,000.

Effect of Salvarsanized and Neosalvarsanized Serum.
T h e problem here was to determine the effect of the living b o d yu p o n the drugs when the latter were introduced into the blood circulation. I t has been assumed t h a t in the animal or h u m a n b o d y t h e y
are converted into highly spirocheticidal substances, as is said to be
the case with respect to the organisms of syphilis, yaws, and relapsing
fevers. I t has already been shown t h a t these drugs are practicallyw i t h o u t effect in vivo u p o n the course of the icteroides infection, andt h a t they are highly destructive to the organism in vitro. I t was thisdiscrepancy which suggested the s t u d y of the salvarsanized serum.

394

ETIOLOGY O~' YELLOW FEVER.

XII

The mode of experiment was to inject a rabbit intravenously with salvarsanor neosalvarsan in a ratio of 0.05 gin. per kilo of body weight, the blood being
drawn 1 hour after the time of injection. Two rabbits, weighing 1,500 and 2,000
gin. respectively, were injected on March 18, 1920, intravenously, one with salvarsan (alkaline), the other with neosalvarsan. After 1 hour they were killed
for the blood. The clear serums were collected the next day and used in theactive state in order to determine whether they were in any way different froma normal rabbit serum when mixed in vitro with the same quantity (1 cc. in eachcase) of a rich culture of Guayaquil Strain 5. The mixtures were kept in a thermostat at 28°C. during the period of observation. Table X gives the results.

The experiments demonstrated clearly the difference between thenormal and the salvarsanized rabbit serums. All the leptospiras mixed
with the latter died within 72 hours, while those in normal rabbit
TABLE X.

Effect of Salvarsanized, Neosalvarsanized, and Normal Rabbit Serum in Vitro.
March 18, 1920.
Dura-

Salvarsanized rabbit serum, !

Neosalvarsanlzed rabbit serum,

Normal rabbit serum, 1 cc.,tlon ofcontact.

cc., plus culture, 1 cc.

I cc., plus culture, I cc,

plus culttue, I cc.

hT$.
1
18

48
72

All active.
All active.
For the most part motile, For the most part sluggish.
but sluggish.
No observation.
No observation.
All dead and degener- All dead and degenerated.
ated.

All active.

No observation.
All active and m u l t i plying.

serum steadily multiplied. It may be mentioned that the reactionsof the drugged serums as well as the reaction of the normal serum
were pH 7.4, hence the question of hydrogen ion concentration does
not enter into the present comparative study. At the end of 18hours the organisms were already less active in the drugged serums
than in the normal serum.
It occurred to me that the cause of this slow leptospiricidal actionof the salvarsanized and neosalvarsanized serums might be due toa gradual development of injurious substances by slow oxidation of
the drugs. If this were the case we should find in these tubes a
powerful and rapidly acting leptospiricidal substance at the end of
72 hours of exposure to the same experimental conditions. Two
different experiments were carried out to ascertain this point.

395

H I D E ¥ 0 NOGUCS~I

In the first experiment 0.5 ce. of a rich culture of the Merida strain1~of icteroides
was added to each of the three tubes containing salvarsanized, neosalvarsanized,
and normal serum respectively. The results were slm~l~r to those observedwith the Strain 5 culture; that is, there was no effect upon the organisms during
the 1st hour; they were all active at the end of that time. After 24 hours theleptospiras in the salvarsanized serum were all dead, but there were many active
survivors in the neosalvarsanized serum. Mter 48 hours, however, they were
for the most part dead in the neosalvarsanized serum, but all were active in the
tube containing the normal serum. In the second experiment three tubes containLug 1 cc. of each of the serums were placed at 28°C. for 72 hours, then 1 cc. of
the rich Merida culture was added to each. The mixtures were again put at 28°C.
for the period of observation. Table XI gives the results of this experiment.
TABLE X I .

Effect on Leptospira icteroides (Mer~ta Culture) of Salvarsanized, Neosal~arsanized,
and Normal Rabbit Serums after the Serums Had Stood far 7g Hours in
tke Incubator at gs°c. *Experiment of Mar.
22, 1920.

After 1 hr.
" 18 hrs.
gg

48

cg

Salvarsanized serum, Nensalvarsanlzed s e r u m ,1 cc., plus culture, I cc. . 1 cc.,
culture,________~lplus¢c.
All active.
Some active.
All dead.

I

All active.
Many active.
All dead.

Normal serum, 1 cc.,plus culture, 1 c c .
All active.
~c~c* There was a slight shift of pH value (to pH 7.8) in these serums on standing,but they were brought back to pH 7.6 by the addition of the culture.
T h e s e two series of experiments indicate t h a t a certain antagonistic
substance seems to h a v e developed in the tubes containing the salvarsanized and neosalvarsanized serums during the period of 72
hours a t 28°C., as shown b y an earlier d e a t h a n d degeneration of the
leptospiras t h a n was the case with the fresh samples of these serums.
T h e difference was especially definite in the effects observed a t theend of 18 hours w i t h the first series. On the other hand, in no instance
was there a n y r a p i d {mmobilization or destruction of the organisms,
the drugs h a v i n g exerted no perceptible effect u p o n t h e m even afteran h o u r ' s contact. A t all events, no rapidly leptospiricidal substances
could be d e m o n s t r a t e d in the salvarsanized or neosalvarsanized r a b b i t
s e r u m after exposure to the air for 72 hours a t 28°C. T h e d e a t h ofthe leptospiras in these drugged serums was slow b u t certain.
X2This strain was isolated from a case of yellow fever in Merida, Mexico,
and will be described in a later paper (Noguchi, H., and Kligler, I. L, J. Exp.
Med., 1920, xxxii, in press).

396

ETIOLOGYO F YELLOW F E V E R .

XIIThe question arises as to the form in which salvarsan or neosalvarsan existed in the blood serum of these two rabbits. This we
do not know, but whatever its state, its ultimate concentration mustcorrespond at most with a dilution of 1:20,000; that is, on the assumption that 0.05 gm. of the compound had diffused out in a spaceof 1,000 cc. In reality, the volume representing i kilo of body weightof the rabbit must be considerably smaller than 1,000 cc., hence theconcentration of the drugs in the serum must have been stronger
than 1:20,000. Other experiments (Table VII) showed that salvarsan or neosalvarsan added directly to a rich culture kills the latterwithin 24 hours; that is, in less time than that required with thesalvarsanized or neosalvarsanized serum, which was at least 48 hours.
Perhaps the arsenic compounds had undergone a modification in the
animal body which converted them into substances operating much
more slowly. As the animals did not urinate after the injection of
the drugs up to the time of collecting the serum the slowness of action
cannot be explained by elimination of the drug through the urine.
Moreover, a dilution of 1:200,000 of the drugs when added directly
to a culture caused the death of the latter in 18 hours. To summarize,
then, serum drawn from rabbits at the end of 1 hour from the timeof an intravenous injection of salvarsan o~ neosalvarsan in a ratioof 0.05 gm. per kilo of body weight possesses a slowly acting leptospiricidal property, which does not seem to be much increased in
respect to rate of action by an exposure to the air for a period of 72
hours at 28°C.

Contrasted Effect of Anti-icteroides Immune Serum in Vitro.
Comparison has already been made of the chemotherapeutic valueof salvarsan and neosalvarsan and the serotherapeutic value of antiicteroides immune horse serum, showing the comparative inefficacyof these drugs and the highly potent specific protective propertyof the serum. Since the leptospiricidal power of the drugs was considerable in vitro, in sharp contrast with their lack of perceptible
protective action in vivo, a similar comparison of the action in vitroof the immune serum and that of the drugs was of practical interest.
The same immune serum which was used in the experiments recorded in Table
V was mixed with icc. of a rich culture of Guayaquil Strain 5, the object being

397

HIDEYO NOGUCHI

to determine how high a dilution of the serum still had leptospiricidal power inthe test-tube. The maximum dilution which caused complete immobilizationand subsequent degeneration within 1 hour was I: 20, while a 1: 200 dilution caused
considerable but incomplete agglutination and degeneration within 18 hours.
No effect whatever was perceptible in a mixture containing the serum in a dilution of 1:1,000 or less. The results of the experiment a~e recorded in Table XII.
W h e n we place side b y side this low titer in vitro of the i m m u n eserum (1 cc. of a 1:100 dilution to 1 cc. of culture) and its protective
titer in vivo (1 cc. of a 1:10,000 dilution to 0.5 cc. of culture, or 5,000m i n i m u m lethal doses), it is easy to conceive at once how completely
TABI, E X I I .

E~ect of Anti-icteroides Immune Serum upon Leptospira icteroides in Vitro.
'March 19, 1920.
Antl-lcteeoidcs immune serum.

After 1 hr.

Mter 18 hrs.

Dilution.

Amount.
CC.

1
1

1:10
1:100

(final dilution 1: 20).
( ""1: 200).

1:1,000 ( "1:10,000 ( "1:100,000 ( "Saline control"""

1:2,000).
1: 20,000).
1:200,000).

All dead.
All degenerated.
For the most For the most part
agglutinated andpart active.
degenerated, b u t
some still motile.
All active.
All active.
gg¢¢

reverse is the relation t h a t exists between the behavior of the i m m u n eserum on the one hand and t h a t of salvarsan and neosalvarsan on
the other toward Leptospira icteroides in vitro and in vivo.
SUMMARYAND

CONCLUSIONS.

I n several series of experiments guinea pigs were variously infectedwith different a m o u n t s of Leptospira icteroides, either in the form ofculture, organ emulsion from infected guinea pigs, or a mixture of
both. T h e infecting materials were of different grades of virulence;in some series the a m o u n t given was near a single lethal dose, inothers a subminimum lethal dose was given, i.e. causing mild infection

398

ETIOLOGY O1~ Y E L L O W I~EVER.

XlI.

with recovery in the majority of animals, and in still others the animals were injected with at least 50 minimum lethal doses of a mixtureof a culture and a highly virulent organ emulsion from a guinea pig.
The animals were inoculated intraperitoneally, and within about 30
minutes each was injected subcutaneously with a different amount ofsalvarsan or neosalvarsan. The amounts injected were in mostseries 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, and 0.03 gin. per 350 tot
450 gun. of body weight, and in one series, in addition to this dosage,
0.00005, 0.0001, and 0.0002 gin. were also tried.
Among the guinea pigs treated either with salvarsan or with neosalvarsan there were more recoveries than among the controls, but t h e ywere not in strict proportion to the amounts of the drugs injected.
In the experiments with 50 minimum lethal doses of the infecting
material there were several recoveries among those which received
0.001 to 0.002 to 0.003 gin., but all passed through a typical infection
with all its symptoms. It is extremely doubtful, therefore, whether
salvarsan or neosalvarsan mitigated the severity of the infection.
The fact is noteworthy that in the same series of experiments theguinea pigs receiving 0.00005 and 0.0001 gm., or thereabout, of salvarsan died 1 to 2 days sooner than the controls, which died in 6 toy
7 days. This suggests a possible earlier injury of the kidneys b y
the drugs, giving the teptospiras an easier and earlier access to, andlocalization in this organ. The ineiiicacy or dubious therapeuticvalue of salvarsan and neosalvarsan against the experimental icteroides
infection of guinea pigs presents a close analogy to the observations.
already made by several investigators with Leptospira icterohoemor-

rhagiee.
Several series of test-tube experiments were also made to determine
the direct effect of salvarsan and neosalvarsan on Leptospira icteroides
cultures. It was found, the injurious effect of alkalinity being eliminated, that the leptospiras remain motile for at least 1 hour in a concentxation weaker than 1:10,000 of salvarsan or 1:1,000 of neosalvarsan. But they become gradually sluggish and succumb to theeffect of the drugs at the end of 18 to 24 hours. The highest dilution
which killed the leptospira in 18 hours was somewhere near 1: 200,000.
When added to a culture medium, salvarsan and neosalvarsan bothsuppressed the growth of icteroides when their concentration in theHmv.YO ~oGucm

399

medium was 1: 200,000. Hence these two drugs are highly poisonousfor Leptospira icteroides.
The serums derived from rabbits which received 0.05 gin. of salvarsan or neosalvarsan per kilo of body weight 1 hour before bleeding
proved to be very different from a normal rabbit serum in their
behavior toward Leptospira icteroicles. In the salvarsanized or neosalvarsanized serums the leptospiras remained active for at least 1
hour but appeared somewhat sluggish at the end of 18 hours, andwere all dead and degenerated when examined after 48 hours. On
the other hand, the leptospiras mixed with normal rabbit serum lived
well and multiplied during the same period of time and under otherwise identical conditions (at 28°C.) To these tubes another portionof culture was added to determine whether or not a rapidly detrimental toxic substance had appeared in the drugged serum while
standing for 72 hours, but the organisms remained still active at theend of 1 hour, 24 hours being required to kill them. In another experiment the salvarsanized and neosalvarsanized serums, together withnormal serum as a control, were first left standing for 72 hours, after
which period a rich culture of icteroides was introduced. The organisms remained uninfluenced for 1 hour in all the serums, but at theend of 24 hours many of those in the drugged serums were dead, andnone was left alive at the end of 48 hours. In normal serum theysteadily increased in numbers and were all active.
It is evident, then, that salvarsan or neosalvarsan introduced intravenously into the body of the rabbit is present in some form in
the blood serum drawn at the end of 1 hour. The substance present
in such serum has a slowly operating injurious effect upon Leptospiraicteroides. The action of the drugs seems to be slower after passage
through the animal body than before. If this phenomenon were to
take place also in the infected body injected with these drugs, it isobvious that in a rapidly evolving infectious disease like yellow fever
the progress of the infection will be too rapid to allow the drugs toexert their beneficial effect upon the course of the disease.
In direct contrast to the behavior of salvarsan and neosalvarsan
in vivo and in ~itro, anti-icteroides immune horse serum in a dose of
0.0001 cc., or 1 cc. of a 1:10,000 dilution, protected guinea pigs froman infection with at least 5,000 minimum lethal doses of icteroides

400

ETIOLOGY

OF Y E L L O W

FEVER.

XII

when injected simultaneously, but the same serum failed to exert
any injurious effect upon the organism when mixed in ~itro in a con~entration weaker than 1: 2,000. A rapid disintegration resulted witha concentration of 1:20 and almost complete agglutination anddegeneration in 1: 200.
The contrast between chemotherapy, as carried out with salvarsan
and neosalvarsan, and serotherapy demonstrated with an immune
serum is apparently of considerable practical significance.