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Journal of Experimental Medicine

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IMMUNOLOGICAL STUDIES WITH A STRAIN OF LEPTOSPIRA ISOLATED FROM~ A CASE OF YELLOW
FEVER IN MERIDA, YUCATAN.
BY HIDEYO NOGUCHI, M.D., AND I. J. KLIGLER, PH.D.

(From the Laboratories of The Rockefeller Institute for Medical Researck.)(Received for publication, April 3, 1920.)

Morphology, Cultural Properties, and Virulence of the Strain ofLeptospira Isolated in Merida.
The morphological features and cultural properties of the strainof Leptospira icteroides isolated in Merida are similar to those of theGuayaquil strains? With respect to pathogenicity and virulence
we obtained the following data.
Guinea Pig 112 Was inoculated on January 4, 1920, with the mixed
emulsions of liver and kidney from Guinea Pig 43, which had typicalsymptoms as a result of inoculation of a culture made with blooddrawn from Case 2 on December 19, 1~19. No. 112 came down withtypical symptoms On the 8th day and was killed for transfer onJanuary 12. The liver and kidney were emulsified together with 0.9
per cent saline solution, about 1 gin'. of organ material to 10 cc. ofsaline solution. The emulsion was allowed to stand several minutes
until the supernatant fluid was freefrom coarse tissue particles. The
clear portion, which under the dark-field microscope showed a few
leptospiras in every field, was used to determine the minimum lethal
dose for guinea pigs. The procedure consisted in inoculating intraperitoneaUy graduated amounts of the emulsion into as many guinea
pigs as the number of dilutions required, each amount being containedin a uniform volume of 1 cc. of 0.9 per cent saline solution. The
results of the experiment are recorded in Table I.
The period of incubation varied from 3 to 7½ days, and deathoccurred 7 to 10 days after the time of inoculation. The durationt Noguchi,H., Y. Exp. Med., 1919, xxx, 13.
627

628

IMMUNOLOGICALSTUDIESONYELLOW

FEVERof illness, calculated from the beginning of fever until death, variedf r o m 2½ to 4½ days. W e did n o t succeed in finding the m i n i m u mlethal dose, which a p p a r e n t l y lies below 0.0001 cc., the smallest
q u a n t i t y of emulsion e m p l o y e d in the p r e s e n t experiment.
T h e M e r i d a strain showed a m a r k e d p a t h o g e n i c i t y for y o u n gpups, 6 weeks old, when given i n t r a p e r i t o n e a l l y 3TABLEI.

Determination of the Minimum Lethal Dose of the Merida Strain.
Guinea pig No.

Amount of organemulsion.

Length of incubation.

Time of death.

CG.

daysdays

163
164
165
166
167
168

0.5
0.2
0.1
0.01
0.001
0.000133½3½S
7~57878109Dog 1.--Jan. 14, 1920. Inoculated with 4 ec. of the kidney emulsion from
Guinea Pig 102 (second passage in guinea pigs) intraperitoneally. The temperature was 39.6°C. on the 4th day, falling rapidly to 38° the next morning.
In the afternoon of the 7th day the animal was found near collapse (37°) andwas killed for examination. Jaundice had been noticed in the eunjunctiva: on
the 6th day, and there was general jaundice on the 7th day. Albuminuria
began on the 5th day and increased until the animal was killed.
Aut~psy.--General icterus; marked congestion of the lungs and other organs.
Hemorrhagic loci were found in small number in the lungs and intestinal mucosa.
The kidneys were congested and degenerated. The liver was mottled withyellowish patches. No subcutaneous or intramuscular hemorrhages were found.
The serous membranes were free from hemorrhage.
Dog 2.--This animal received the same material as Dog 1 at the same time.
The temperature curve was somewhat more irregular, 39°C. on the 2nd and 3rddays, normal on the 4th day, and 39.6° on the 5th day. The decline was very
rapid, 37.5° a.m. and 36.2° p.m. on the 6th day. The animal died during thenight. Albuminuria was present on the 4th day. Jaundice was noticeable on
the 5th day and had definitely increased during the 6th day.
Aut~psy.uGenexal jaundice; liver and other organs yellowish. Few small
hemorrhagic loci in the lungs. The stomach contained some digested blood,s Villamil Mendoza, M., Apuntes acerca de la fiebre amarilla, Thesis, Merida,1920.

629

HIDEYO NOGUCHI AND I. J. KLIGLER

and the mucosa of the intestines was profusely hemorrhagic. The kidneys werecongested and degenerated. Subcutaneous tissues, muscles, and serosa nothemorrhagic.

Identification of the Merida Strain and the Therapeutic Value of
Anti-icteroides Serum against This Strain.
T h e i d e n t i t y of the Merida strain of the leptospira was establishedb y means of a series of experiments in which varying quantities ofanti-icteroides serum were injected, together with 1 cc. of an emulsionof the liver and kidney from Guinea Pig 43, into several normal
TABLE II.

Ident*'ficaaon of the Merida Strain and the Therapeutic Value of Anti-icteroides
Serum against This Strain.
GuineaNo.pigAmountofserum.

109
110
111
112
113
114
115

0 (control).
0( " ).
0( " ).
0.1
0.01
0.001
0.0001Result.

Lesions.

CC.

Killed after 6 days.
Died " 6 "Killed " 4 "Survived.

Typical,"lung lesions.

guinea pigs. T h e anti-icteroides serum h a d been prepared in a horseb y repeated injections of different strains of Leptospira ideroides
from Guayaquil cases. T h e results are recorded in T a b l e II.
T h e outcome of this experiment m a d e it evident t h a t the Merida
strain belongs to the same group as the Guayaquil strain.
P o l y v a i e n t anfi-ieteroides immune serum prepared in the horse or
m o n o v a i e n t anfi-icteroides i m m u n e serum prepared in the rabbit hada definite devitalizing action upon the Merida strain, while antiserums similarly prepared with strains of icteroh~emorrhagice had noperceptible effect upon the Merida strain.

630

IMMUNOLOGICAL STUDIES ON YELLOW ]FEVER

Serotherapeutic Experiments.
The purpose of the next series of experiments was to ascertain
whether or not the same immune serum possessed a therapeutic value
in infection with the Merida strain such as had been demonstrated
in the case of experimental infection in guinea pigs with the Guayaquilstrains? Thirty-one guinea pigs were inoculated at the same timewith 0.5 cc. of the organ emulsion, representing at least 5,000 minimum lethal doses. 1 hour after inoculation two of the animals weregiven an injection of 0.0001 cc. of the serum, and two more received
an injection of 0.1 cc. The procedure was continued with each groupof four animals after periods of 24, 48, and 72 hours and 4 and 5 days
respectivelyl After 5 days the amounts of serum injected wereincreased to 1 and 2 cc., as it had been found that the smaller doses
had no effect at this period of the disease.
All the animals which received no serum until after 72 hours showed
fever at that time; in those untreated until 4 days there were feverand slight jaundice; those not treated until 5 days showed a decline
in temperature and increasing jaundice; and in the group which
received the injection after 6 days there were intense jaundice andsubnormal temperature, and the animals were near or in collapse.
The results of this series are summarized in Table III.
0.1 cc., therefore, of the anti-icteroides serum prevented any external
manifestation of the infection if given before the onset of fever (within
72 hours after inoculation). The same dose, given to animals in thefebrile stage, b u t without jaundice, prevented the development ofjaundice; animals still having fever and showing more or less jaundiceat the time of injection of this dose likewise survived, and in somejaundice rapidly disappeared in 24 hours. On the other hand, thisquantity of serum failed to prevent a fatal outcome when given toanimals in which defebrescence and increasing jaundice had set in.
The nephritic symptoms, which had existed since 72 hours afterinoculation, were rapidly increasing b y this time.
0.0001 cc. of the serum prevented a fatal infection in three out offour guinea pigs when given within 24 hours. In one animal whicht Noguchi, H., J. Exp. Med., 1920, xxxi, 159.

HIDe.Y0 NOGUCHI AND I. J. KLIGLER

631

died from the typical infection there was a prolongation of the incubation period (6 days) and the duration of illness (5½ days). In
two of the three surviving animals fever developed after 4 days, butthey recovered without showing jaundice at any time. Injection ofthis quantity of serum 48 hours after inoculation, or later, had noeffect upon the course of the disease, all animals so treated dying
within 5 to 8½ days with typical symptoms.
Seven guinea pigs which were near or in a state of collapse 6 days
after the inoculation were given 1 cc. (three animals) and 2 cc. (fouranimals), but with one exception all died within ½ to 2½ days from thetime of injection of the serum.
It may therefore be concluded that the anti-icteroides serum here
employed is able, in a dose of 0.1 cc., to protect a guinea pig from afatal infection against at least 5,000 minimum lethal doses of theMerida strain when administered during the incubation period or atany early stage of the disease (fever and beginning of jaundice), butthat it has almost no effect upon the course of _the disease if given at
a later period when the animals are deeply yellow and the temperature
has begun to go down. At or near collapse even 1 or 2 cc. failed toprevent death in the majority of animals. On the other hand, aquantity as minute as 0.0001 cc., when given within 24 hours, is able
to protect the majority of animals from a fatal infection.
If man's degree of susceptibility to Leptospira icteroides is comparable to t h a t of the guinea pig, it may reasonably be assumed thatthe injection of the anti-icteroides serum at an early period of thedisease will have a beneficial effect similar to that observed in thetreatment of the experimental infection in guinea pigs. On the basisof body weight a man of 80 kilos would require about 200 times theamount of serum needed to save a guinea pig of 400 gm.; that is, 20cc. (0.1 cc. X 200 = 20 cc.). The mode of administration should
be intravenous, and if necessary the injection should be repeated atshort intervals (4 hours).

632

IMMUNOLOGICAL S T U D I E S ON YELLOW ~ E V E R

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634

IMMUNOLOGICAL STUDIES ON YELLOW FEVER

Pfeiffer Phenomenon with the Serums of Yellow Fever Convalescentsin Merida.
Pfeiffer's phenomenon with Leptospira icteroides had been previously
observed with a limited number of serums from yellow fever convalescents in Guayaqull 4 and was positive in about 85 per cent of thecases studied. It was desirable that this line of observation should
be extended to yellow fever cases existing elsewhere, and we availed
ourselves of the present opportunity with the Merida cases. Our
first intention was to study as many specimens of serum from yellow
fever convalescents in Merida as could be obtained, with as m a n ystrains of cultures of icteroides as possible. This plan could not be
carried out, however, for two reasons; there were only a few convalescents accessible, and all the Guayaquil cultures brought downby us to Merida were lost within a few days after our arrival, andbefore our work had been started, by an accidental rise of temperaturefor 3 days to 56°C. of the thermostat in which the cultures had beenplaced for safety. Hence work on the Pfeiffer phenomenon waspossible only after the isolation of Leptospira icteroides from Case 2.
There were two convalescents at the yellow fever hospital (Casade Salud), Cases 2 and 3. 5 Both patients (soldiers) were infectedin the same house in Merida, Case 3 preceding Case 2 by perhaps 2weeks. The serum for the Pfeiffer test was drawn at the end of the5th week of the disease in Case 3 and during the 3rd week in Case 2.
Both specimens were still distinctly jaundiced. Through the courtesyof Dr. A. Lara we were given specimens of serum from two otherconvalescents who had had a typical attack of yellow fever in August,1919; that is, about 5 to 6 months previously. One was a youngwoman, whose ~arine still contained albumin at that time, but who
showed no bile pigment either in the urine or in the serum.
The following procedure was employed. 0.5 cc. of each specimen ofserum was mixed with 1.5 cc. of a pure culture of the Merida strainof the leptospira and the whole ~njected into the peritoneal cavityof a guinea pig. The exudate was withdrawn from the peritoneal
cavity after 30 minutes for dark-field examination. Table IV is arecord of the results.
4 Noguchi, H., J. Exp. Med., 1919, xxx, 9.
s Noguchi, H., Y. Exp. Med., 1920, xxxii, 601.

HIDEYO NOGUCHI AND I. J. KLIGLER

635

The serums from yellow fever convalescents in Merida gave uniformly a positive Pfeiffer reaction when tested with a culture of theleptospira isolated from Case 2. Although the guinea pig inoculatedwith the mixture of this culture and the serum from the same patientdied of secondary infection too soon to permit any observation withregard to the power of the serum to protect the animal from a fatal
infection, the animal which received the serum from Case 3 with thisculture escaped the infection altogether. It may be supposed that
this patient had been attacked by the same strain that later infected.
the other two soldiers (Case 1, fatal, and Case 2) living in the same
TABLE IV.

Pfeiffer Reaction with Serum from Convalescents and the Merida Strain.
SerumNo.

.Tlrpeof
infection.

Leng~
time
onset
dise~

ffir

Pfeiffer
reaction.

Remarks.

wks

1 (Case 2).
2( "3).

3 (civilian).

4(").

No serum(control).

Moderatelysevere.
Modemtelysevere.

3Typical.

5Positive,5"NTegatiw

Died of intercurrent infection within
24 hrs.
No infection resulted.

Died after 7 days with typical infection.
Died after 7 days with typical infection.
Died after 9 days with typical infection.

house, hence such complete protection. On the other hand, Serums
3 and 4 were from two civilians who had recovered from yellow feverat least 5 months previous to the time when their blood was tested.
These serums gave a positive Pfeiffer phenomenon but failed toprotect the animals.
These four serums were brought back to The Rockefeller Institute
to be tested with the Guayaquil strains of Leptospira ictertrides andalso with some of the strains of Leptosplra icterol~morrhagit~. The
serums from Cases 2 and 3 were accidentally lost during transportation. Moreover, during the journey the remaining two had to be636

I M M U N O L O G I C A L STUDIES ON Y E L L O W FEVEI~

kept at a comparatively high temperature (heated steamer cabinand trains) for 15 days, and their activity must have suffered considerably. Nevertheless, tests were made on March 5; that is, 2months from the time of collection. The amount available of Serum
3 was 0.75 cc. and that of Serum 4, 0.9 cc. Each specimen wasdivided into three equal parts and tested with two Guayaquil icteroides
strains (Nos. 1 and 5) and with the American No. 1 strain of icterohcemorrhagi~e. The results obtained are presented in Table V.
T A B L E V.

Pfeiffer Reaction with Serum from Convalescents Tested with Two Guayaquil Strainsof Leptospira icteroides and One Strain of Leptospira icterokcemorrhagio,~
Leptospira icteroides.

Le#tos#ira iaeroka~morrkagim.

Serum No.

34
N o serum

Guayaquil Strain 1.

Guayaquil Strain 5.

American Strain I.

Positive.
Partial reaction.
Negative.

Doubtful.
Partial reaction.
Negative.

Negative.
~c(control).

While no definite conclusions can be drawn from the results obtainedwith these old serums, it is possible to recognize an unmistakablespecific reaction with the icteroides strains, particularly between Serum
3 and No. 1 of t h e Guayaquil icteroides strains. With Serum 4 there
was not complete destruction of the icteroides organisms; but about
one-half of them became paralyzed, and some were degenerated,
although many active organisms were simultaneously present. There
was no effect whatever upon American Strain 1 of Leptospiraicterohcemorrhagice.
SUMMARY.

Identification of the leptospira isolated from a case of yellow feverin Merida was accomplished by means of an anti-icteroides immune
serum prepared in a horse with several Guayaquil strains of Leptospiraicteroides. The immune serum showed a protective action of hightiter against the Merida strain, thus establishing its efficacy as atherapeutic agent against this strain. Polyvalent anti-icteroides

HIDEYO NOGUCHI AND I. J. KLIGLER

637

immune serum prepared in the horse or monovalent anfi-icteroides
immune serums prepared in the rabbit had a definite devitalizing
action upon the Merida strain, while immune serums similarly prepared with strains of icterohcemorrhagia~ had no perceptible effectupon the Merida strain.
Serums from yellow fever convalescents in Merida gave a positive
Pfeiffer reaction with the Merida strain of Leptospira icteroides. The
reactions between the Guayaquil strains (Nos. 1 and 5) and two ofthese serums from convalescents varied "from definitely positive todoubtful, owing probably to the diminution of active immune principles in the serums during the prolonged and unfavorable conditionsof their transportation.