野口英世著

Journal of Experimental Medicine

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I M M U N O L O G Y OF T H E P E R U V I A N STRAINS OF L E P T O SPIRA I C T E R O I D E S .
BY HIDEYO NOGUCHI, M.D., AND I. J. ICLIGLER, ProD.

(From the Laboratories of The Rockefeller Institute for Medical Researck.)(Received for publication, August 7, 1920.)
In the course of a study of the etiology of yellow fever in Peru, I
an effort was made to solve several related problems of the disease.
The first point taken up was the behavior of the serums of Peruvian
convalescents in Pfeiffer's reaction towards strains of Leptospiraicteroides isolated elsewhere; the second Concerned the action, in
vitro and in vivo, on the Peruvian strains of the leptospira, of a polyvalent immune serum prepared in horses with several strains of
Leptospira icteroides of Guayaquil origin; the third related to thedifference in the natural resistance to the leptospira infection of thenative guinea pigs as compared with guinea pigs recently broughtfrom New York; and the fourth and last point bore on the availabilityof old rabbit serum fo~ cultivating Leptospira icteroides. The lasttwo questions had arisen as a result of the rather unfavorable outcome of the work carried out in Payta, and, as will appear later,proved to be important.

Pfeiffer Reaction with Serum from Convalescents.
The method was similar to that previously employed. 1 cc. ofeach serum was mixed with 0.2 cc. of a rich 6 week icteroides cultureof Guayaquil Strain 1 and the Merida strain. The guinea pigs usedwere those recently brought from New York. Observations weremade at the end of 1 hour. ~1 Noguchi, H., and Kligler, I. J., y. Exp. Med., 1921, xxxiii, 239.

This work was done at the Belan Hospital, Piura, where we received cordial
cooperation from Dr. M. Guzman R., Dr. A. Gonzalez, and Dr. Prieto, as well as
the mother superior. We also wish to thank especially Dr. Marcos L. Vega, thegovernment sanitary officer at Piura, for the arrangement to test tho eonvales,cents in Piura.
253254PERUVIAN

STRAINS O F L E P T O S P I R A ICTEROIDES

Nine convalescents were accessible for this test. Of these, fourcame from the Payta (1920)i four from the Piura (1919), and one
from the Morropon (1920) epidemic. The serums were obtainedin the five 1920 cases within a period ranging from 7 to 36 days fromthe time of onset of the fever and in the four 1919 cases at the end of
10 to 11 months. The results obtained are shown in Table I. Theinterpretation of the findings is evident. All the Peruvian cases,
except two instances to be' discussed below, gave a positive Pfeiffer
reaction to two different strains of Leptospira icteroides, irrespective of whether the case occurred in Payta, Piura, or Morropon.
T A B L E I.

Pfeiffer Reaction with Serum from Convalescents.
Serum No.

Locality ofepidemic,Length of time between onset ofyellow fever andwithdrawal ofblood.

Strain ofLeptospiraicteroides.

Results.

daysPayta.
c~

7736
17

G u a y a q u i l No. 1~c~c1Positive.

c~mO$,Piura.
~ci1
101010Merida.
gg
ccgcDoubtful.
Partial reaction.

da~
9Control.

Morropon.

21Positive.
Negative.

The exceptions were the serums obtained from two persons who had
had yellow fever 10 months previous to the time of testing, bothgiving a slight or partial reaction. It may be noted, however, thattwo other individuals who had had an attack 10 and 11 months
previously reacted positively. The reason for a partial reactionin the first two cases probably relates to a gradual diminution of theantibodies responsible for the Pfeiffer reaction.
The foregoing observations are of value in establishing the identityof the organisms that were present in individuals suffering fromyellow fever in Ecuador, Mexico, and Peru. The positive Pfeiffer

255

HIDEYO NOGUCHI AND I. J. KLIGLERreaction with the serums of yellow fever convalescents in Payta
also indicates that the guinea pigs of the P a y t a experiments which
had shown suspicious febrile reactions and hemorrhagic lesions of thelungs and kidneys without developing a typical fatal infection, had
actually suffered a mild infection with L e p t o s p i r a ictvroides. Finally,it may be added that the guinea pigs which were employed in thePfeiffer tests and reacted positively showed no symptoms and survived, while the control animal (inoculated with the Merida strain)
suffered from a typical but non-fatal infection.
Virulence.

The virulence of the Morrgpon strains was next tested on New
York guinea pigs. The material used was an emulsion of kidney
TABLE II.
Determination of the Minimum Lethal Dose of Morropon Strains.
Amount of kidney emulsionin 1 ec.

StrainL
Incubation
period.

Result of inoculation.

Incubation period.

Died in 9 days.

No test made.
343550.1
0.01
0.0Ol
0.00Ol0.00001443½
55½

Result of inoculation.

daysdays

1Strain 3.

Recovered.
Died in 11 days.
No infection.

No test made.
Died in 8 days.

"" 10 "

The animals used for this experiment served at the same time as controls forthe therapeutic experiments reported in Table III.
made in saline solution in a ratio of approximately 1 gm. to 10 cc.
After the coarse tissue fragments had settled, the turbid supernatantportion was employed for inoculation. Ascending tenfold dilutions
were prepared with 0.9 per cent Saline solution, and 1 cc. was injected
intraperitoneally into guinea pigs, one animal being used for eachdilution. Table II gives the minimum lethal dose as determined forMorropon Strains 1 and 3. The approximate minimum lethal dosefor Strain 1 lies between 0.0001 and 0.00001 cc., and that of Strain
3 beyond 0.00001 cc. The grade of virulence of the strains is, therefore, about the same as that of strains previously studied.

256

P E R U V I A N STRAINS OF LEPTOSPIRA ICTEROIDESTherapeutic Experiments with Anti-icteroides Serum.
The reaction of the Peruvian strains towards the anti-icteroides
immune serum prepared with the Guayaquil strains confirms theother evidence of the identity of the organisms. It has been shownin our first paper 1 that the leptospira from Peru gave a positive
Pfeiffer reaction with this immune serum. In the present series ofexperiments the object was to determine, in guinea pigs given multiple
minimum lethal doses of Morropon Strains 1 and 3,. the prophylactic
and curative values of a specific polyvalent anti-icteroides immune
horse serum. The immune serum was administered in varying
quantities and at different periods after inoculation, even after theappearance of symptoms, up to the time when the animals were fast
approaching the terminal stage of the disease induced. The guinea
pig kidney emulsions used for infecting the animals were injected
intraperitoneally in uniform amounts of 0.2 cc., this dose representing
at least 2,000 minimum lethal doses of Strain 1 and 20,000 minimum
lethal doses of Strain 3. The immune serum was given intraperitoneally also in doses of 0.00001, 0.0001, 0.001, 0.01, and 0.1 cc.,contained in saline solution to a total volume of 1 cc., and finally
1 cc. of undiluted serum. In order to obviate the factor introduced
by an occasional unusually refractory animal, more than two of
the recently brought guinea pigs were used in testing the effect ofeach dose of serum. Table III, which summarizes the results,
brings out the following points. (a) The anti-icteroides serum iscapable of checking the development of the infection, provided asufficiently large quantity is given during the incubation period.
The four deaths which took place among animals treated during
this period occurred among those which did not receive serum until
72 hours after inoculation; i.e., at the end of the incubation period.
(b) A quantity of serum larger than 0.01 cc., given during the febrile
stage (96 hours after inoculation) usually prevented the infection fromreaching the icteric stage. This was true particularly with the animals infected with Strain 1, which had scarcely a tenth of the virulenceof Strain 3. (c) The effect of the serum became uncertain when itwas given during the icteric stage (5 to 6 days after inoculation).
With 0.01 cc. no~ animal was saved, while 0.1 cc. saved three out of

257

HIDEYO NOGUCHI AND I. J'. KLIGLER

four infected, with Strain 3, but none with Strain 1. 1 cc. of undiluted
serum saved one out of three with Strain 1 and three out of four with
Strain 3. That the proportion of recoveries among the animals
treated during the icteric stage does not correspond with the virulenceof the strains can be explained only on the basis of the natural resistance of individual guinea pigs; at all events, death occurred inboth groups, even when 1 cc, of serum was given.
TABLE HI.

Protective and Curative Effect of Anti-icteroides Serum in Guinea Pigs Experimentally Infected with Leptospira icteroides.
Animals treated before beginning offever.

Strain.

kmount ofserum.

0.2 cc. of Strain 1culture, representing 2,000 minimum
lethal doses.

0.0001
0.001
0.01
0.1
10.2 cc. of Strain 3
culture, representing 20,000 minimum lethal doses.

0.0001
0.001
0.01
0.1
12544Animals treated dur- Animals treated aftering'~ebrile stage.
jaundice appeared.

1000353053430001202205323322002230012440330T h e controls w i t h o u t serum t r e a t m e n t are represented b y the a n i m a l s of t h eexperiment recorded in T a b l e II.

Resistance of Native and Imported Guinea Pigs to Leptospira icteroides.
Certain tests made with guinea pigs obtained in Payta suggested
that the native Peruvian guinea pigs are more refractory to the
leptospira than guinea pigs brought from New York. To determine
this point four groups of guinea pigs were inoculated, on May 16,
1920, with a uniform amount of kidney emulsion from a guinea, pig
which had shown a typical infection with Morropon Strain 4. The

258

PERUVIAN

STRAINS

OF LEPTOSPIRAICTEROIDES

animals inoculated comprised: (1) fifteen native guinea pigs which
had once been inoculated with culture materials in Payta, without
any definite infection having been induced; (2) six native guinea
pigs which had not been used; (3) eight American guinea pigs which
had formerly been inoculated with culture materials in Payta, without
infection having been induced; and (4) four newly imported Americanguinea pigs. Table IV summarizes the result. It will be noted thatthe virulence and dose of the strain used were such that even theyoung American guinea pigs, used as a standard of susceptibility,
did not all develop a fatal infection. This effect was rather anadvantage, since, had multiple fatal doses been given, the differencein susceptibility might not have come out so clearly.
TABLE

IV.

Relative Resistance of Native and American Guinea Pigs to Leptospira icteroides.

Guinea pigs.

Native (onceused). . . . . . . . . . . . . . . . . . . . . .
"(normal) . . . . . . . . . . . . . . . . . . . . . . . .
American (old lot; once used) . . . . . . . . . . . . .
"(new lot; n o r m a l ) .

EscapedTotal No. infection(noinjected.

Animals showing
definite infection.*jaundice).

15684Died.

9630* This includes guinea pigs which showed typical lesions when killed.
The striking feature of the foregoing experiment is the complete
resistance of all six normal native guinea pigs in contrast to thefour normal American animals, of which two, at least, died of a typicalinfection. Among the animals inoculated on a former occasion,both the native and the American groups contained susceptible aswell as refractory ones, the difference being that a larger proportionof the native (60 per cent) than of the American (37.5 per cent) proved
resistant.
The relatively large proportion of resistant animals among thenative guinea pigs, as well as among the American guinea pigs which
had withstood the unfavorable climate and other hardships, is apoint to be taken into account in future work.

HIDEYO N O G U C H I AND I. J. K L I G L E R

259

Deterioration of Rabbit Serum through Age.
That an alteration takes place in rabbit serum when it is subjected
to long transportation in a hot climate is evident from the fact thatno growth of Leptospira icteroides could be obtained with such serum.
On the other hand, old rabbit serum which has been kept in therefrigerator up to 6 weeks has been found still suitable for making
subcultures of icteroides. Whether the same would be true after 2
or 3 months standing at 4°C. has not, however, been determined.
The rabbit serum which was used in the cultivation experiments in
Payta I was 3 months old and had been exposed to the ordinary
tropical temperature of the region, which is sometimes 40°C. during
the day, and it contained considerable precipitate. It was thoughtprobable that the deterioration of the serum through age and climaticconditions might have been a factor in the unsuccessful outcome of
the Payta experiments. Moreover, another lot of serum, which had
been collected in New York in April and hermetically sealed, wasbrought down to Piura under ordinary temperature conditions.
Some tubes contained precipitate, while others had remained clear.
It was used when 50 days old for making subcultures of four Morropon strains of the leptospira, but when the culture tubes were
examined 10 days later no growth could be found. Fearing a possible
loss of all strains, we made two more sets of subcultures of the samestrains, one set with the old and another with fresh rabbit serum.
With the media containing the fresh rabbit serum a rich growth took
place with three of the four strains, but there was no growth in thetubes prepared with the old serum.
Other factors, however, made it difficult to determine how important It is in general to employ fresh serum in culture work. withicteroides. The Peruvian strains were unusual in that they early
degenerated in culture. The initial cultures from human cases never
became very rich, and they disappeared within about 3 weeks. The
first generation cultures from the infected guinea pigs grew fairly
well within a week, .but at the end of 2 weeks examination showed
only a few active organisms and many degenerating. Of the culturesof Strain 3, none was alive, and injection into guinea pigs failed torecover it. The remaining strains (Nos. 1, 2, and 4) were successfully

260

P E R U V I A N STRAINS OF L E P T O S P I R A ICTEROIDES

subcultured by using fresh rabbit serum, as noted above. It iscertain that all the strains would have been lost if we had relied
entirely on the old serum. The present study does not indicate
whether the importance of using fresh serum applies only to theprimary human and recently isolated generations or even to remotesubcultures.
SIYM'MAR¥.

Serum from yellow fever convalescents from Payta, Piura, andMorropon gave a positive Pfeiffer reaction with the strains of Leptospira icteroides isolated in Guayaquii and Merida. The serum also
protected the guinea pigs from these strains in the majority of instances. The Pfeiffer reaction was complete with all recent convalescents (7 to 36 days) but slight or partial in some instanceswith serum derived from individuals who had had the attack ofyellow fever 10 months previously.
The virulence of the Morropon strains was found to be approximately the same as that of the Guayaquil or Merida strains. With
one strain the minimum lethal dose for the guinea pig was less than
0.00001 cc. of a kidney emulsion from an infected guinea pig.
Suitable quantities of the anti-i~teroides serum administered toguinea pigs inoculated with 2,000 to 20,000 minimum lethal doses ofinfective material prevented the development of the infection, ora fatal outcome, according as the serum was given during the incubation period or after fever had appeared. The earlier the administration of the serum the smaller was the quantity needed; during
the incubation period 0.0001 to 0.001 cc. was sufficient, during thefebrile period 0.01 to 0.1 cc. was required to check the progress ofthe disease, and even at the time when jaundice had already appeared,
the injection of 0.1 to 1 cc. saved three out of four animals inoculatedwith Strain 3 and one out of three inoculated with Strain 1.
The native guinea pigs secured in Payta proved to be unusuallyrefractory to infection with Leptospira icteroides as compared withnormal guinea pigs recently imported from New York.
Fresh rabbit serum is recommended for culture work with Lepto-spira icteroides.