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Journal of Experimental Medicine

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ETIOLOGY OF YELLOW FEVER.
X. COMPARATIVE IMMUNOLOGICAL STUDIES ON LEPTOSPIRA ICTEROIDES AND LEPTOSPIRA ICTEROHeEMOR_RHAGI~.
BY HIDE¥O NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Researck.)(Received for publication, November 18, 1919.)

In a previous paper 1 it was reported that serum from convalescentyellow fever patients has a more or less marked antagonistic effectupon Leptospira icteroides derived from certain cases of yellow feverin Guayaquil, as manifested by a positive Pfeiffer phenomenon in
the peritoneal cavity of the guinea pig. In a few instances the serum
protected the guinea pig from a fatal infection with the organism.
A similar result was obtained with the serum of guinea pigs wl~ch
had recovered from a non-fatal experimental infection with theleptospira.
In the present paper the question of immunity will be more fully
discussed, particularly with regard to agglutination, lysis, complement fixation, Pfeiffer's reaction, etc., with immune sera preparedin rabbits and horses by repeated inoculations of Leptospira icteroides.
Experiments have also been carried out to determine the relation
between this organism and Leptospira icterohcemorrhagie~of infectious
jaundice by means of cross-immunity reactions in vitro and in vivo.
In the in vivo experiments not only passivel but also active immunity
has been taken into consideration. A part of this study has alreadybeen published in a paper dealing with the leptospira isolated fromwild rats and mice in Guayaquil 2 and will not be repeated here.
t Noguchi, H., Y. Exp. Med., 1919, xxx, 9.
Noguchi, H., J. Exp. Med., 1919, xxx, 95.
135

136

ETIOLOGYOF YELLOW

FEVER.

X

Production of Immune Sera.
Monovalent immune sera for each of four strains of Leptospira icteroides were prepared in rabbits by injecting the animals intravenouslywith 2 to 4 cc. of rich live culture, on rabbit serum medium, several
successive times at intervals of 7 to 14 days. The animals showed adefinite febrile reaction on the 4th or 5th day after the first injection
but no other symptoms. Subsequent inoculations produced noperceptible reaction in most of the animals, although some diedsuddenly, probably owing to the development of anaphylaxis as thenumber of inoculations increased.
Polyvalent immune serum was produced in a horse by injecting
intravenously (jugular vein) gradually increasing amounts of rich live
cultures (horse serum medium) of five strains (Nos. 1, 3, 4, 5, and 6)
of Leptospira icteroides. 20 cc. of the mixture of cultures were given
as the initial injection, and subsequent injections were increased up
to 200 cc. This dose was maintained for most of the time during
immunization. The first inoculation caused a rise in temperature
to 40°C. on the 3rd and 4th days, with considerable swelling along
the inoculated side of the neck. The animal lost its appetite during
the period but regained its normal condition within 5 days. There
was no jaundice at any time. Subsequent inoculations caused noperceptible reaction. During a period of 65 days the horse received
2,495 cc. of mixed live cultures in fifteen injections. The following
protocol gives the schedule of immunization.

Horse 2.--Feb. 6, 1919, 20 cc.; Feb. 11, 40 cc.; Feb. 15, 60 cc.*;Feb. 19, 125 cc.;
Feb. 24, 150 cc.; Mar. 1,200 cc.; Mar. 6, 200 cc.; Mar. 11, 200 cc.; Mar. 15, 200cc.; Mar. 19, 200 cc.; Mar. 24, 200 cc.; Mar. 28, 200 cc.; Apr. 2, 200 cc.; Apr. 7,
200 cc.; Apr. 12, 300 cc. First bleeding on Apr. 19, 19i9.
Effects of Immune Sera upon Leptospira icteroides and Leptospiraicterohcemorrhagie,.
Monovalent immune sera were prepared in rabbits, as described,
and experiments conducted to demonstrate the effects of such sera
upon homologous and heterologous strains of Leptospira icteroides
on the one hand and of Leptospira icteroha~morrhagia~ on the other.

HIDE¥O NOGUCHI137

The experiments were designed to throw more light on the relationthat m a y exist, not only among different strains of these organisms,
but also between Leptospira icteroides and Leptospira icterohcemorrhagice as distinct varieties. The effects of an immune serum are
manifold, b u t w e confined our observations to the phenomena of
agglutination, immobilization, and disintegration of the organism
when mixed with the immune serum in vitro and that of the reactionof Pfeiffer following the simultaneous inoculation of the organism andan immune serum into the peritoneal cavity of guinea pigs. Comp.lement fixation tests were also made.
Agglutination.--The technique employed for the agglutination
tests was as follows:
Rich live cultures, grown at 26°C. for 2 to 3 weeks on rabbit serum medium(one part of serum plus three parts of 0.9 per cent sodium chloride solution), of eachstrain were selected. 1 cc. of each%f a number of cultures was put into a series ofsmall sterile test-tubes, and 0.2 cc. of the fresh immune serum to be tested wasadded to each tube. Controls with normal rabbit serum accompanied each seriesof experiments. The culture and immune serum were carefully mixed by gentle
shaking and the tubes incubated at 37°C. (water bath) for 2 hours. Examination~of the contents were then made by means of the dark-field microscope.
With pronounced agglutination minute particles could be macroscopically detected. Another examination was made on the tubes
after they had been left at room temperature for 96 hours, b u t theresults were identical with those recorded after 3 hours, with thepossible exception of a more granular appearance of some of theagglutinated cultures and in extreme instances a thin b u t definite
grayish sediment at the bottom of the test-tubes.
In the present series of experiments there were available threemonovalent antisera for Leptospira icteroides, comprising Strains 1, 5,
and 6, and six for Leptosplra icteroheemorrhagice, comprising theJapanese, British, American No. 1, Group 8, Group 11, and Group 30
strains. Against each of these nine monovalent immune sera were
tested cultures of five different strains of Leptospira icteroides, Nos.
1, 3, 4, 5, and 6, and seven strains of Leptospira icterohcemorrhagiee,
Japanese, British, French, American No. 1, Group 8, Group 11, andGroup 30.

138

E T I O L O G Y OF Y E L L O W ]?EVER.

X

A strong immune serum acting upon the homologous strain of

Leptospira icieroides agglutinated the organisms quickly into rather
large masses, in which they appeared tightly held together. :Mostof the organisms became immobile, gradually lost their elementary
windings, and were soon transformed into stiff, irregularly, granularfilaments. If the serum was not strong enough to produce thesechanges, the agglutinated masses were for tile most part degenerated,
but with several apparently intact immobilized organisms a t tile
periphery. In other instances the agglutinated masses contained
a certain number of individuals which were still active, while instill others the organisms in agglutinated masses showed quivering
motility in some part of their body. On the whole, agglutination is
the first and more constant reaction observed and disintegration thesecondary and less constant (Tables I and n).
Pfei•er'sPhenomenon.--To complete, the observations, Pfeiffer
tests were also perfolwned with the immune sera and the various
strains of icteroides and icterohcemorrhagice. The technique employed
was that generally followed. 1 ec. of a given immune serum was
mixed in a Petri dish with 1 cc. of a rich live culture and immediately
injected into the peritoneal cavity of a normal guinea pig. The
peritoneal fluid was withdrawn with a capillary pipette after 30
minutes and 2 hours and examined under tile dark-field microscope(Tables I and n).
The reaction of Pfeiffer with the immune sera and homologous
strains of Leptospira icteroides is prompt and complete. The organisms seem first to be agglutinated into large masses and then to bequickly disintegrated. The phenomenon m a y be complete within
15 minutes, so that no trace of the organisms can be seen in theperitoneal fluid. The same is true of Leptospira icterohcemorrhagiceand the homologous immune sera. A decided increase of actively
motile organisms was noticed in the guinea pig peritoneal cavity
when a normal rabbit serum instead of a specific immune serum
was used.
As shown in Table I, the five different strains of Leptospira icteroides
reacted to each of the three monovalent immune sera. The intensityof agglutination and disintegration varied somewhat according towhether the strains were homologous or heterologous. Without

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142

E T I O L O G Y OF Y E L L O W F E V E R .

Xexception the strongest reaction was obtained with the homologous
and a less pronounced one with heterologous strains. Disintegrationof the organisms was usually complete in the homologous but seldom
so in heterologous combinations. Normal rabbit serum exerted
neither an agglutinating nor a disintegrating influence upon any of
the strains studied. On the contrary, the addition of normal rabbit
serum to control tubes kept the organisms active for many days.
That these anti-icteroides sera did not agglutinate the various strainsof Leptospira icterohcemorrhagice to any marked degree is also shownin this table. There were a few instances in which slight agglutination was observed, b u t none so marked as that which occurred withthe strains of icteroides. In contrast to the results obtained with theicteroides strains, in no instance was there any disintegration of theicterohcemorrhagice organisms by an anti-icteroides serum. The Pfeiffer
phenomenon was invariably positive with the anti-icteroides serum
and the ieteroides strains, b u t almost always negative when the antiieteroides serum was tested with the ieterohcemorrhagice strains. In
two instances there Was a suggestive reaction.
Table II presents the results obtained with six different monovalent anti-icterohcemorrhagice sera. The marked difference thatexists between the icteroh6emorrhagice strains and the icteroides strains
is shown. Aside from slight variations, the icteroh6emorrhagice strains
reacted with the icterohcemorrhagice antisera quite generally andstrongly and present a marked contrast to the icteroides strains,which reacted occasionally and never very strongly. The Pfeiffer
phenomenon was positive in all combinations of the anti-ieteroh~emorrhagice serum with the icterohcemorrhagice group, b u t only occasionally and slightly with the icteroides group. The occurrence of afairly marked agglutination in certain instances, such as, for example,
anti-Japanese serum versus No. 1 icteroides strain or anti-group No. 11
serum versus No. 3 icteroides strain, is of considerable interest becauseof the occurrence of similar weak reactions among the combinationsof anti-icteroh¢emorrhagice sera and certain heterologous icterohcemorrhagice strains. The only reliable differentiation in these instances
would seem to be that of the Pfeiffer test. Beyond these few irregularities the behavior of the imrhune sera towards icterohcemorrhagiceand icteroides seems to warrant the conclusion that t h e strains of

HIDEYO N o o v c m

143

Leptospira icteroides and Leptospira icteroh~emorrhagice form closely
related b u t distinct groups.

Complement Fixation.--The technique used in the c o m p l e m e n t
fixation tests was as follows:
Rich cultures of various strains of Leptospira icteroides and Leptospira icterokemorrhagice were grown on rabbit serum media, killed by placing the culture
tubes in a water bath at 60°C. for 10 minutes, and then used as antigens in thecomplement fixation tests. Graduated quantities (0.1, 0.05, 0.02, 0.01, 0.005 co.)of each of the monovalent sera were mixed with a uniform quantity of the antigen(a quantity which had been found to be non-anticomplementary in several doses
when tested with 0.1 cc. of complement), and to each tube was added 0.1 cc. offresh guinea pig serum as complement. The mixtures were brought up to a total
volume of 1.5 ec. for each tube by the addition of 0.9 per cent saline solution.
After incubation at 37°C. for 1 hour, 0.1 cc. of a 20 per cent suspension of washedsheep corpuscles and 0.1 ce. of anti-sheep amboceptor (rabbit serum), representing
three hemolytic units, were added to each tube, the contents thoroughly mixed,
and once more incubated at 37°C. for 30 miuntes. The results were read after
standing for 1 hour. The actual quantity of each antigen used was 0.1 cc. of thekilled culture, which exhibited only a slight anticomplementary property whenused alone in quantities of from 0.4to 0.6 c o .
I n the m a j o r i t y of instances the reaction was m a x i m u m with 0.1 cc.
of i m m u n e serum, and all tests with 0.005 cc. gave a negative result.
T h e readings of the reaction obtained with 0.1 cc. of the antigens and
0.1 cc. of the i m m u n e sera are recorded in T a b l e I I I .
Complete fixation took place when the i m m u n e sera were mixed
with the homologous strains, b o t h in the case of Leptospira icteroides
and in t h a t of Leptospira icterohcemorrhagiee. Occasionally, especiallyamong the icteroides strains, the fixation was as strongly positive with
one or the other of the heterologous strains as with the homologous.
T h e r e were a n u m b e r of instances also in which a more or less definite
fixation occurred when the anti-icteroides sera were mixed with theicterohcemorrhagi~e strains or the anti-icterohcemorrhagice sera with theicteroides strains, some reactions being as strong as +. Generally
speaking, however, there was only a limited degree of cross-reaction
between icteroides and icterohcemorrhagice.
Variations in the fixation reaction were r a t h e r m a r k e d among thedifferent strains of icterohcemorrhagice, according to the combinationsof the i m m u n e sera and heterologous strains, some of which showed
only a feeble fixation ( + ) with certain sera.

144

ETIOLOGY OF YELLOW FEVER.

X

TABLE III.

Complement Fixation Tests.
Strain ofL,pto.1 Strain of ~pto@~midero-

spire ia~roldes.

Serum.

Immune serum
Strain 1.

for

Leptospirc~ icteroides,

Immune serum
Strain 5.

for

Leptosp~ra icteroides,

Immune serum
Strain 6.

for

Leptospire

iaeroides,

Tmmune serum for Leptosplra iaeroh~norrhagie, Japanese strain.

Immune serum for Leptospira iaerotumnorrlmg/w,"British strain.

Immune serum for Leptospira iaerotuemorrlmgice, American Strain I.

Immune serum for Leptospira icterolumnorrhv
giw, Group 8 strain.

145

t~u~EYO NOGUCHITABLE III---Concluded.
Strain of Lep~

Strain of Leptosptr,,ia~o-Serum.

Immune serum for Leptosplra iaerokcmorrkag/C, Group l l strain.

Immune serum for Leptospira ¢cterolavraorrkag/~, Group 30 strain.

++i++++++
i lll

+++
++,

* Four plus signs vertically placed indicate no hemolysis, or complete fixation;
three plus signs, about 25 per cent hemolysis; two plus signs, about 50 per centhemolysis; one plus sign, about 75 per cent hemolysis. ~- indicates almost complete hemolysis, and minus, complete hemolysis, or no fixation.

Protective Properties of Immune Sera against Leptospira icteroides.
Mono~alent Immune Rabbit Sera.--Several monovalent immune
rabbit sera, some specific for the icterddes strains and some for theicterohcemorrhag¢~, were tested for their protective properties against
the experimental infection in guinea pigs with icteroides or with~terohcemorrhaglce. For this purpose t cc. of culture of the strain to
be tested was mixed with 1 cc. of immune serum and the mixture
injected intraperitoneally into the guinea pig (Table IV).
I t was found that Nos. 5 and 6 of the icteroides strains wereprevented from causing any infection in guinea pigs when inoculated
simultaneously, not only with their respective antisera, but also in
combination with heterologous antisera. This would indicate thatan immune serum prepared with one strain of Leptospira icteroides
is equally protective against another strain of the same organism,
T h a t the protection was specific may be seen from the fact that these
two strains of Leptospira icteroides caused either a fatal or a rather

146

ETIOLOGYOF

YELLOW

FEVER.

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148

ETIOLOGYO F YELLOW F E V E R .

Xsevere infection in guinea pigs when mixed with normal rabbit
serum or with one or another of the antisera produced with different
strains of Leptospira icterohcemorrhaglce. It is noteworthy that theperiod of incubation when anti-icterohcemorrhagice serum and Leptospira icteroides are combined is somewhat prolonged. For example,
with Strain 5 of icteroides the control animMs with normal rabbit
serum died in 6 or 7 days, while the guinea pigs receiving the antiict~rohcemorrhagice sera died between 9 and 11 days after the inoculation. This is also true of Strain 6, with which death occurred in 6days in the control animal and in 10 and 12 days in the animals
injected with the anti-lcterohcemorrhagice sofa. The symptoms andlesions were typical in all instances in which an infection ensued.
The results obtained by reversing the combinations, that is by
mixing different strains of Leptospira icterohcemorrhagice with antiict, roides immune sofa, show also an unmistakable specificity of theprotection afforded by these immune sofa. A clear-cut specific protection is shown in the experiments with the Japanese and Guayaquil
strains of icteroheemorrhagice; these three strains were effectively
neutralized by their homologous immune sera, but not by any antiicterMdes immune sofa, although the sera seemed to delay death insome instances. The French strain of icteroh¢emorrhagi~ewas least virulent and did not cause fatal infection in guinea pigs when any one of
the immune sera was simultaneously inoculated. The examinationof the surviving guinea pigs for the lesions, particularly those in thelungs, after 24 days showed that the guinea pigs which received theanti-icteroides sera had numerous old hemorrhagic loci in the lungs,
while none or only a few loci Were found in those which were inoculated with the anti-lcteroh~morrhagie sofa.
Polyvalent Immune Horse Sera.--A horse was immunized with amixture of cultures of four strains of Leptospira icteroides for a periodof 65 days, during which 2,495 cc. of the cultures were injected intravenously as described in the protocol above. At the same time
another horse, which had once been immunized for a period of several
months in 1918 with various strains of Leptospira icterohemorrhagice,
was injected again, with icterohcemorrhagice cultures, comprising ninestrains: Japanese (one strain), American (three strains), French(one strain), British (one strain), and Guayaquil (three strains).

HIDEYO NOGUCIffI149

The serum from each horse was then tested for its protective property
in guinea pigs against some of the representative strains of Leptospiraicteroides and Leptospira icterohcemorrhagice.
The procedure consisted in injecting into the peritoneal cavity ofguinea pigs a mixture of a given quantity of culture (or liver emulsion)of a strain with 1 cc. of immune serum, either full strength or diluted,
as indicated in Tables V and VI.
For each dose of the serum two guinea pigs were used inorder to determine its titer as closely as possible. Only one guinea
pig, however, was used in determining the protective titers of theanfi-icteroides serum against the icterohcemorrhagiee strains or viceversa. The amounts of culture (or liver emulsion) used in testing
the corresponding antiserum were such as to approximate 500 minimum
lethal doses, while for a cross-titration usually about 50 minimum
lethal doses, or even 10 minimum lethal doses were chosen. The
reason for reducing, the number of minimum lethal doses in crossprotection experiments was that in several preliminary tests a larger
quantity of culture was found not to be influenced to any great extent
by a heterologous antiserum.
The anti-¢cteroides serum protected guinea pigs against approximately 500 minimum lethal doses of Leptospira ¢cterMdes in doses of
0.001 (for Strain 5) and 0.0001 cc. (for Strain 6). In other words,
1 cc. of this serum neutralizes about 500,000 to 5,000,000 minimum
lethal doses of Leptospira icteroides, according to the degree of virulence of the culture. On the other hand, at least 1 cc. of the same
serum was required to protect guinea pigs against 10 to 50 minimum
lethal doses of the Japanese and French strains of Leptospira icteroheemorrhagi~e. 0.1 cc. of the serum averted a fatal outcome but
fail¢d to prevent wholly the infection. The difference in the protective
efficacy of the anti-icteroides serum against icteroides and icterohcemorrhagiee is striking.
The anti-icteroheemorrhagiee serum exhibited also a marked specificprotective property for the Leptospira icterohcemorrhagice strains,neutralizing at least 5,000,000 (for the French) to 500,000 (for theJapanese) Tn~nlrnum lethal doses per 1 cc. Its effect upon the icteroides strains was in each case feeble but distinct, since it protectedguinea pigs completely against 50 minimum lethal doses per 1 cc.

150

ETIOLOGY OF YELLOW FEVER.

X

TABLE V.
Protective Properties of Polyvalent Anti-icterdd, es Serum against Leptospiraicteroides and Leptospira icterotu~morrlmgice.
Strain of Leptosplraicteroide.s.
Anti-/d~d&sserum.

No. 5.
O. 1 CC. Of culture(about 500 u. L. D.).

Strain of Lcptos~tra icterol~raorrluggica.

Japanese.
No. 6.
0.I cc. of liver emul0.1 cc. of culture(about 500 M. "~. D.). sion(about I0 ~ . L . v . ) .

French.
0.I cc. of culture(about 50 M. L. D.).

$$.

Died in 7 days. Died in 7 days.
Typical symptoms and lesions.
" " 10 "Died in 9 days.
Typical symptoms and lesions.

Died in 7 days.

Died in 9 days.

0.00001
0.00001

Survived.
Died in 9 days.

Died i n , d a y s .

Died in 10 days.

0.00010.0001

Survived (I).

Died in 8 days.

Survived.

0.000001

0.000001

t~

tt. 9

g¢

" " 9 "
Survived.

0.001
0.001

Died i n l 4 days. Died in 13 days.

0.01
O.O1

Died i n l 0 days. Died in 13 days.

0.1
0.1

Survived.
fever.

Had Survived.
fever.

Had

1.0
1.0

Survived.
fever.

Had Survived.
fever.

Had

Normal horse Died in 7 days. Died in 7 days. Died in 7 days. Died in 8 days.
serum 1.0 cc. Typical symp- Typical symp- Typical symptoms and letoms and le- toms and ]e(control).
sions.
sions.
sions.
a n d p r e v e n t e d d e a t h , b u t n o t i n f e c t i o n , w h e n u s e d i n a d o s e of 0 . !
cc. I t will b e seen, t h e r e f o r e , t h a t t h e r e s p e c t i v e p o l y v a l e n t a n t i s e r a
e x e r t a p o w e r f u l a n ~ i h l ] a t i n g effect i n g u i n e a p i g s u p o n t h e i r c o r r e s p o n d i n g t y p e o r g a n i s m s , b u t t l i e r e also e x i s t s a n u n d e n i a b l e , t h o u g hHIDEYO NOGUCHI

151

feeble, cross-protective reaction, which m a y be explained by assuming that the two groups of organisms are not altogether alien but
are closely related to each other; they may even constitute two
subspecies or races.
TABLE VI.

Protectire Properffes of Poly~alent Anti-iaerotavmorrhagic~ Serum against
Leptospira ixterohvemorrkag~e and Leptospira iaeroides.
Anti-~aeroImmorrkag~
serum.

Straino f Lepgospira icteroh~moml~gi~.

Straino f Leptosplra iavroides.

Japanese.
French.
No. 1.
0:1 cc. of culture
Icc. of culture
0.1 cc. of culture(about 500 t~ T. 9.). (about 500 ~. T,.D.). (about 50 M. T.,m).

No. 5.
0.01 cc. of culture(about 50 ~. L, 9.).

Died in 6 days.

CC.

O. 000001

0.000001
0.00001
0.00001"" 11 "

Died in 10 days.
" " 13 "Died in 8 days.

Survived (t).

Survived.
Died in 11 dgys.

Died in 7 days.

Died in 8 days,

Survived.
"

Died i n 8 days.

Died in 8 days.

Died in 7 days.

Died in 9 days.

Died in 8 days.

Died in 15 days.

Survived; fever.

Survived; mild
jaundice.

Survived.

Survived.

Died in 6 days.

Died in 7 days.

0.00010.0001

Survived.
Died in 8 days.

0.001
0.001

Survived.

"

g~

C¢

0.01
0.01
0.1

¢¢

g¢

0.1

g~

g¢

1.0
1.0

g¢

g¢

Normalhome serum 1.0 cc.

Died in 7 days.

Died in 7 days.

(control).

Active Immunity.
Guinea pigs vary considerably in their susceptibility to Leptospiraicteroides. I t sometimes happens that a culture which kills guinea
pigs of average susceptibility in a dose of about 0.0001 cc. occasionally

152

ETIOLOGY OF YELLOW ]?EVER.

X

fails to produce a fatal infection in a guinea pig in a dose as large as
0.01 or 0.1 or even 1 cc. Such refractory guinea pigs are rarely met
with, b u t the fact that there exist certain unusually resistant indivlduals is of great interest. I t has also been found that there are
exceptionally susceptible individuals which respond to infection withan attenuated culture which no longer attacks t h e average guinea
pig. For example, with certain cultures of Leptospira icteroides
which had been repeatedly subcultured without passage through theguinea pig for many months, only one out of severalan~rnals inoculatedwith the same culture m a y come down with typical symptoms. In
fact, when the first attempt to restore the virulence of the culture b yanimal passage failed, a second or third attempt with four or fiveguinea pigs each time was necessary to obtain a single positive result.
These facts furnish possible explanations for certain paradoxical
results which are sometimes encountered in determining the state of
immunity.
In a discussion of active immunity we m a y distinguish between
that which arises from recovery from a genuine infection and thatwhich follows the inoculation of killed organisms. Animals which,after receiving an inoculation of a sublethal dose of a live culture,
do not react definitely, may acquire a state of immunity similar tothat of vaccinated animals.
The results with the guinea pigs which had recovered from a moreor less pronounced infection after the inoculation of a culture or blood
derived from a guinea pig dying of the typical infection with Leptospira icteroides will first be described. The tests consisted in thePfeiffer phenomenon and the effect of the inoculation of different
cultures in case of a negative Pfeiffer reaction.

Series 1.
Four guinea pigs were actively immunized with Strain 5 of Leptospira ¢cteroides.
Guinea Pig Ch/.--Nov. 27, 1918. Received 1 cc. of citrate blood from a guinea
pig which had been infected with Strain 5 of Leptospira icteroides and showed thetypical symptoms of icteroides infection. After the usual course of infection(fever, slight icterus, albuminuria) the animal became well within 2 weeks. Dec.
19. Received 1 cc. of culture of the same strain, No symptoms followed. Jan.
6, 1919. Received another injection of culture of the same strain, without any
perceptible effect.

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153

Pfd.ffer Reacgon.--Jan. 18. A rich culture of the same strain (1 ce.) was injected intraperitoneally. A prompt positive reaction was obtained. Jan. 22.
The Pfeiffer reaction was tested with Strain 6, 1 cc. of a rich culture being used.
Result positive. On the same day, 4 hours later, another Pfeiffer test was madeon this animal with the Japanese strain of Leptospira $ctgrohemorrhag¢~, 1 ce. ofculture being used. Result negative. This animal died in 23 days with thetypical symptoms of ideroluemorrlmg~ infection.
Guinea Pig Ch 2.--The procedure with this animal was similar to that with
Guinea Pig Ch 1.
Pfeiffer Reaction.--Jan. 18, 1919. Tested with 1 co. of culture of the samestrain. Result positive. Jan. 22. Pfeilter test with 1 cc. of Strain 6 culture waspositive. The same day, 4 hours later, tested with 1 cc. of French strain of Leptospira icteroturmorrhagize. Result negative. This animal died in 14 days withthe typical symptoms of icterol~morrkagia~ infection.
Guinea Pig Ck 3.--The procedure with this animal was similar to that with
Guinea Pig Ch 1.
Pfdffer Reaaion.--Jan. 18, 1919. Test with 1 cc. of the same strain was positive. Jan. 22. Test with 1 cc. of the British strain of Leptospira icteroheemorrhag/~ was doubtful. The animal survived.
Guinea Pig Ch 4.--The procedure with this animal was similar to that with
Guinea Pig Ch 1.
Pfeiffer Reaaion.--Jan. 18, 1919. With 1 cc. of culture of the same strain test
was positive. Jan. 22. With 1 cc. of culture of Strain 6 of Leptospira ictertrides
test was positive. The animal survived.
Series 2.
Three guinea pigs, out of a large number inoculated with Strain 6 of Leptospira icleroides, which suffered more or less severe infection and eventually recovered, were reinoculated with a culture of the same strain once or twice (as described in the protocols below) afterwards, but showed no characteristic symptoms,
except a rise of temperature for a day in one (Guinea Pig C 2). Hence they werecompletely immune to the same strain and were used to test their resistance tocertain other strains, including the Japanese strain of iaerohoemorrhagice.
Guinea Pig C/.--Dec. 3, 1918. Received 1 cc. of Strain 6 culture of Leptospiraicteroides. The animal had typical fever and a trace of jaundice, but recovered inabout 9 days. Two more injections of 1 cc. of the same culture were given, one
on Dec. 19, and another on Jan. 6,1919.
Pfeiffer Reaction.--Jan. 18. With 1 cc. of a rich culture of the same strain, aprompt and positive reaction. Jan. 22. Inoculated with 1 cc. of a rich cultureof Strain 5 of Leptospira icterMdes. Pfeiffer reaction positive. No symptoms
within 1 month.
Guinea Pig C 2.--Dec. 11, 1918. Received 1 cc. of blood from a guinea'pig
showing typical symptoms (fever and slight jaundice). The animal eventually

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ETIOLOGY OF YELLOW FEVER.

X

recovered. Jan. 6, 1919. 1 cc. of a rich culture of the same strain was given.
There was slight fever for a day, but no infection followed.
Pfeiffer Reaction.--Jan. 18. 1 cc. of a culture of the same strain was inoculated
intraperitoneally, but no organism could be found in the peritoneal exudate after
30 minutes. Jan. 22. The animal was tested again for the Pfeiffer reaction, with
1 cc. of a rich culture of Strain 1 of Leptospira icteroides. There was a complete
positive reaction within 30 minutes, and no infection followed the inoculation.
Guinea Pig C 3.--This animal was immunized in the same way as Guinea Pig
C2.
Pfeiffer Reaction.--Jan. 18, 1919. 1 cc. of a rich culture of the same strain was
injected intraperitoneally. Reaction prompt and complete. Jan. 22. 1 cc. of
a rich culture of the Guayaquil strain, Group 30, of Leptospira icterohremorrkagice
was inoculated intraperitoneally. When examined after 2 hours the organisms
were partially agglutinated, but most of them were actively motile. On the 4th
day the temperature rose and remained above normal for 3 days. There was aslight jaundice on the 9th and 10th days, which soon faded. The animal survived.
In this instance there existed a mild infection with the icterohcemorrhagicestrain.

Series 3.
Nov. 27, 1918. Three guinea pigs were inoculated with a culture of the Japanese strain of Leptospira icterohcemorrhagice. A second injection was given on
Dec. 19, and a third on Jan. 6, 1919. The animals showed a definite but mild
infection after the first inoculation, but recovered. No symptoms followed thesecond or third injection of the same strain.
Guinea Pig J 1. Pfeiffer Reaction.--Jan. 18, 1919. With a culture of theJapanese strain, test positive. The animal remained well.
Guinea Pig J 2. Pfeiffer Reaction.--Jan. 18, 1919. With a rich culture of
Strain 5 of Leptospira icteroides, test negative. The animal survived, passing
through a moderately severe/ctero/des infection.
Guinea Pig J 3. Pfeiffer Reaction.--Jan. 18, 1919. Pfeiffer reaction with arich culture of Strain 6 of Leptospira icteroides not clear-cut. There was a tendency to formation of agglomerated masses, without immobilization or lysis of
the organisms. The animal showed mild but typical symptoms of icteroides
infection after 13 days, but eventually recovered.

Supplementary Experiment with Anti-icterohosmorrhagice Serum.
Jan. 16, 1919. A number of guinea pigs were inoculated with mixtures of a
polyvalent anti-icteroluemorrhag~ horse serum prepared by Ina~a and Ido and different strains of Leptospira icterotmmorrkagice and Leptospira icteroides, with a view
to determining the protective property of this serum against the icteroluemorrkag~
as well as the icteroides strains. 1 and 0.1 co. of the serum were used, mixed with
0.5 co. ~f a culture of each strain, and inoculated at once into the peritoneal cavity

HIDEYO N O G U C H X

155of guinea pigs. The protective titer of the serum had previously been tested
against the Japanese strain of Leptospira icteroh~morrkag~, and the serum had beenfound to neutralize 1 cc. of the culture in a dose of 0.001 cc. The result of thepresent experiment showed that the guinea pigs which received the immune serumand cultures of the Japanese, French, American, and Guayaquil strains of ic~ro/ ~ r r / ~ g ~ z survived, while the control animals without the seruni died in 6, 9, 8,
and 7 days respectively. There were no symptoms observed in the surviving
animals at any time; they were completely protected by the serum in the doses
given (1 and 0.1 cc.). All the guinea pigs inoculated with a culture of the British
strain of ic~roh~zmorrh~g~ with or without (one control) the addition of theimmune serum survived; that is, the culture employed was apparently avirulent.
The result obtained with the strains of Leptospira ict~ro~s was somewhat
surprising. The strains used in this group were Nos. 5 and 6, which killed thecontrol animals in 5 and 10 days respectively Both cultures, however, produced
only a temporary febrile reaction when injected together with 1 ce. of the serum
and a moderately severe but non-fatal infection with 0.1 ce.
All the guinea pigs surviving in this series of experiments were subjected on
Feb. 10, 25 days after the first inoculation, to immunity tests with cultures ofvarious strains. The guinea pig which had received on Jan. 16 the mixture of theJapanese strain and 1 ce. of anti-icterohcemorr~gi¢ serum escaped infection andwas inoculated with 0.5 cc. of a culture of Strain 5 of Leptospira icteroides on
Feb. 10. I t died with typical symptoms of icteroides infection in 7 days. The
guinea pig which escaped infection "with the mixture of the French strain and
1 cc. of a n t i - @ t e r o ~ h a g f z serum on Jan. 16 and was injected with 0.5 cc.
of Strain 5 of icteroides on Feb. 10 died in 5 days with typical symptoms of~teroM~s infection. The guinea pig which escaped infection with the mixture ofAmerican Strain 1 and 1 cc. of anti-@terotus~norrhagfz serum on the first injection
and was inoculated with 0.5 cc. of Strain 5 of icteroi&s on Feb. 10 finally recovered after a moderately severe/ctero/des infection.
As already mentioned, the guinea pigs which were inoculated on Jan. 16
with 1 and 0.1 cc. of anti-icterohcemorrhagix serum, together with the culturesof Strains 5 and 6 of Leptospira ~cteroides, had a texhporary febrile reaction or amoderately severe infection. These animals were injected on Feb. 10, 25
days later, with a culture of the Japanese strain. All except one, which showed
only slight lesions in the lungs, died with the typical symptoms of icterohcemorrhag~ infection in 8 to 9 days. This would seem to indicate that theseanimals were protected by the anti~terohzmorrhag~ serum from the fatal
outcome of the /~tero/des inoculations, but they were not rendered immune
against the Japanese strain of icterokcemorrhagire when tested 25 days afterwards. Also the anti-icterohemorrhagi¢ serum injected on the first occasion
had no perceptible protective action against this strain after a period of 25
days.

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ETIOLOGY 01~ YELLOW :FEVER.
SUMMARYAND

X

CONCLUSIONS.

It has been previously reported* that a filterable microorganism
belonging to the genus Leptospira has been recovered from the bloodor organs of human beings suffering from the disease known as yellow
fever in Guayaquil, and that the organism, which has been termed
Leptospira icteroides, induces in certain experimental animals thecharacteristic symptoms and lesions observed in the patients fromwhom it was isolated. It has also been previously shown I that theserum from patients recovering from an attack of yellow fever in
Guayaquil had the power to agglutinate and dissolve the organism
when introduced into the peritoneal cavity of a normal guinea pig(Pfeiffer phenomenon). Moreover, the guinea pigs which had once
been inoculated with the blood of yellow fever patients without
succumbing to the infection, notwithstanding the fact that they had
shown a definite febrile reaction after 4 to 5 days, were found to be
refractory to a subsequent inoculation of a culture of Leptospiraicteroides. 4 All these observations pointed to the possible relationof this organism to the disease known as yellow fever in Guayaquil.
The demonstration of the filterability of the organism* and thetransmission of the infection with the same organism by Stegomyia
calopus ° have further strengthened the probable etiological significance of the organism in yellow fever.
It was by no means a simple problem to determine the relation existing between Leptospira icteroides and Leptospira icterohcemorrhagie~.
An experiment reported in a previous paper seemed to justify theview that the two leptospiras ~ are closely related but not identical,
yet it was necessary to exhaust various other modes of differentiation
before the distinction between them was firmly established. The
present paper continues this phase of the inquiry in further detail.
There have been taken up here the phenomena of agglutination,
the reaction of Pfeiffer, complement fixation, the protective properties of various monovalent and polyvalent immune sera, and active
8 Noguchi, H., J. Exp. Meet., 1919, xxlx, 547, 565, 585; xxx, 87.
4 Noguchi, H., J. Exp. Med., 1919, xxx, 1.
6 Noguchi, H., J. Exp. Med., 1919, xxx, J3.
6 Noguchi,H., Y. Exp. Med., 1919, xxx, 401.
Noguchi, H., J. Exp. Med., 1919, xxx, 95.

HID~YO l~ooucm

157

immunity. As the result of experiments in connection with these
immunity phenomena the following data are presented.
Monovalent immune sera prepared by several successive injections in an animal naturally refractory to Leptospira icteroides possess
the power to agglutinate in dtro not only the homologous strains, butalso all other strains of icteroides tested. On the other hand, a slighteffect, or none at all, has been observed when these immune sera
have been mixed in vitro with various strains of Leptosp#a icterohcemorrhagloe. A similar relation exists between the monovalent antiicteroh~eraorrhagice sera and the various strains of Leptospira ieteroides; that is, there is a slight agglutinating effect in some instances
upon the icteroides strains, but it is never so strong as that occurringin tests against the icterohcemorrhagiee strains. The Pfeiffer reaction
gave a sharper differentiation between the two groups, for in ~ o s t
instances the phenomenon was specific for the group. There wereoccasional doubtful reactions, but not enough to warrant a confusionof the two groups.
Polyvalent immune sera, one specific for icteroides, and the otherfor icteroh~morrhagice, showed a high titer of neutralizing power forthe cultures of the homologous groups. It was found, however,that the action of the sera is by no means absolutely specific, because
the injection of a sufficient amount of the anti-icteroides serum apparently prevented a fatal outcome in a guinea pig inoculated withmultiple minimum lethal doses of a culture of Leptosp#a icteroheemorrhagiee, and dee versa. The specificity of the serum was demonstrated only when it was used in smaller quantities.
More or less specificity was shown by the complement fixation
reaction, b u t it was not absolute. Weak fixation occurred when theanti-icteroides serum was mixed with one or the other of the icteroheemorrhagia~ strains and vice versa, and strong fixation occurred
only when the antiserum was mixed with one of the icteroiges strains.
The question naturally arises whether or n o t t h i s apparent specificity
is due to the homology of the serum and not altogether to a differencein genus of the strains. In other words, it is justifiable to question
whether all these variations in the degree of intensity of the reaction
are not due to strain variations of the same genus. This question is
not finally settled by the present investigation, in which only four

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ETIOLOGY OF YELLOW FEVER.

X

icteroides and nine icterohcemorrhagia~ strains have been carefully
studied. Nevertheless, on the basis of the findings with these thirteen
strains, it seems probable that Leptospira icteroides and Teptospiraicterohcemorrhagice are closely allied but are nevertheless distinct intheir immunological reactions. Perhaps the difference between thetwo may amount to that between subspecies or races. It has been
pointed out earlier that the pathogenicity of the two is also distinct,
inasmuch as icteroides produces chiefly icterus and nephritis andicterohcemorrhagice hemorrhage and nephritis, the icterus being lessand the hemorrhage more prominent in the evolution of the latter
infection.
In the study of active immunity--exclusive of vaccination-difficulty has been experienced in the evaluation of the results, owing
to the existence of natural resistance to infection among guinea pigs.
A guinea pig may recover from the inoculation of Leptospira icteroides
and then resist a subsequent inoculation with a virulent strain of
Leptospira icterohcemorrhagice, a condition simulating that brought
about by the identity of the two organisms. However, the refractoriness of such an animal to icteroh~morrhagi~e may be due to its naturalimmunity to it. In the present study, therefore, only those guinea
pigs were selected which had reacted typically--though in mild degree- - t o the icterbides infection, in order to determine whether they were
subsequently immune to the inoculation of icterohceraorrhagice, lndeed, by this mode of experimentation it was found that the guinea
pigs which had once passed through an attack of the icteroides infection
were absolutely immune to a second infection with the same organism
but reacted severely and sometimes fataUy to a later inoculation of
iaerohcemorrhagiae. Although there were a number of instances inwhich a previous infection with icteroides did not confer any perceptible
immunity upon the guinea pigs against icteroha~morrhagice, anothergroup of guinea pigs showed a considerable resistance to the icterohcemorrhagia~ infection as compared with th6se which had never been
inoculated with icteroides. There is not much doubt, therefore,that an icteroldes attack brings about, in some instances at least,a certain degree of resistance to the icterohaemorrhagice infection.
Hence the study of the phenomena of active immunity strongly
indicates that icteroides is closely related immunologically to ictero-hcemorrhagiee.