野口英世著

Journal of Experimental Medicine

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ETIOLOGY OF Y E L L O W FEVER.
II.

TRANSMISSIONEXPERIMENTS ON YELLOW FEVER.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
PLATE 35.

(Received for publication, March 27, 1919.)
For the purpose of transmitting yellow fever to animals experiments were made to reproduce the disease in whatever kind o f animal was available by inoculation with the blood from patients admitted to the Yellow Fever Hospital in Guayaquil during my staythere. The blood was drawn from the median basilic vein of the patient at various stages of the disease, mainly during the 1st week,with a sterile Luer syringe and injected before coagulation into theperitoneal cavity (in mammals) or into the pectoral muscles (in birds).
10 cc. of blood were usually taken and distributed among from one
to four animals in each instance. When more than one kind ofanimal was used for one patient correspondingly larger quantities of blood had to be drawn. The following animals were employed: ringtail monkey, rabbit, guinea pig, cat, dog, donkey,
guatusa, 1 comadreja, 2 and ozo-melero ~ among the mammalians;
pigeon, paloma de tierra, 4 bluebird, mantas, 5 blackbird, parrakeet, 6
reedbird, blancos, 7 and diostede 8 among the birds.
1 Dasyproaa aguti (animal akin to rabbit).
2 Bassaris bassaricyon (?) (resembling an opossum).
s Cholopus didactylus (two-toed sloth).

Family of Peristerinm (ground-dove).
Siskin (probably).
6 Family of Bolborhynchus.
Similar to a magpie, but white.
8 Genus Pteroglossus.
565

566

ETIOLOGY OF YELLOW FEVER.

II

All the animals inoculated were kept under observation at least 1
month before being discarded as negative, even when no symptoms
were observed. An animal which showed a rise of temperature
after a period of several days was closely watched, and when morethan one of the animals of the series receiving the same blood became febrile one was killed for examination and further passage madein the same species of animal. As will be seen later (Case 2) such a
step was essential with some specimens of yellow fever material, as
b y this means alone was it possible to reproduce an experimental
condition resembling yellow fever on the third animal passage.
None of the birds showed a n y definite symptoms when first inoculated with the blood of yellow fever patients, but upon subsequent
injection of the organ emulsion of a guinea pig with the experimental
yellow fever 9 parrakeets, mantas, blackbirds, reedbirds, and palo~ade tierras died within 24 to 72 hours. There were no lesions except
f o r hyperemia of the visceral organs. The pigeons, bluebirds, andtoucans showed no definite reaction. This rather rapid death following the second inoculation may have been due to an anaphylactic
phenomenon. Rabbits and guatusas showed a temperature of 40-41°C.
on the 4th to 5th day after inoculation but became normal after 48
hours. There were no other symptoms, and the animals remained
well during the period of observation (1 month). The ringtail
monkeys showed a similar but more prolonged fever reaction lasting
several days. The animals appeared depressed after 48 hours andrefused to take food. The conjunctivae were injected for several
days, but there was no jaundice. Unfortunately the number of ex:periments with this animal was limited to five because of the difficuIty in securing them in sufficient numbers. Donkeys showed noreaction to the injection of yellow fever blood in large quantities.
Apparently none of the animals enumerated above possesses a
sufficient degree of susceptibility to enable the yellow fever virus tomultiply and reproduce the symptoms and lesions known as yellow
fever in man. It is possible that some of them had an extremely
mild or atypical form of the infection.
9 For the sake of brevity the condition induced by inoculation will be termed"experimental yellow fever." Further evidence bearing on the validity of thisterm will be presented in successive papers of the series.

HIDE¥O NOGUCHI

567

Since guinea pigs could be secured more easily, and in view of thesuccess of Inada and Ido ~° with this species in the study of infectious
jaundice, they were extensively used during the present study.
Guinea pigs are native to Ecuador and are found wild in the mountainous regions." All except 60 of the guinea pigs which were usedin these experiments (which were taken from New York) were rearedin the mountains and shipped to the Yellow Fever Hospital. They
stand captivity well for several months.

Experiments with Guinea Pigs.
Blood and Organ Emulsions.~Of 74 guinea pigs inoculated withthe specimens of blood from 27 cases of yellow fever, 8, representing 6
cases, came down with the symptoms resembling human yellow
fever. In one instance, however, the.reproduction of the diseaserequired three successive inoculations in guinea pigs. In this instance one of the animals showed a rise of temperature on the 5th day
and was killed for examination and transfer on the 6th day. The
liver was degenerated, and the kidneys were highly congested. A few
hemorrhagic spots were found in the lungs. In a further passage ofthe organs to two new guinea pigs there was a similar febrile reaction
with ecchymoses in the lungs but only a trace of icterus. On transferof the second passage material typical hemorrhages, icterus, and albuminuria were induced. With the materials from only one of several
autopsies was a positive transmission obtained. The details of thetransmission experiments follow.
The first positive transmission was obtained in the case ofPatient A.
Case 1 (Text-Fig. 1, a).--A. A., female, age 17 years; servant, native of Latacunga (9,055 feet above sea'level). Had come to Guayaquil 3 months previously.
Onset, July 14, 1918. Headache, chills, fever, and severe pains throughout
the body. July 15. Vomited chocolate-colored matter and felt pains in epi10Inada, R., Ido, Y., Hoki, R., Kaneko, R., and Ito, H., ]. Exp. Med., 1916,
xxiii, 377.
tl Guinea pigs are in the market for food in Ecuador, but the municipality of
Guayaquil has forbidden their being kept in the city on account of the possibility of spreading bubonic plague.

568

ETIOLOGY OF YELLOW FEVER.

II

gastric region. July 19. Admitted to hospital. Was very sick and vomited
dark bloody matter. Conjunctiwe intensely injected and distinctly jaundiced.
Liver tender but not palpable. General jaundice of the skin. Blood drawn frommedian basilic vein into citrate solution and inoculated into guinea pigs immediately and also after 3 days cultivation at 25°C. July 20. Patient felt better,but gums began to bleed. Abdominal pains not marked. July 22. Condition
worse. Patient very ill, vomited constantly ("coffee-grounds vomitus"). Jaundice very marked. Liver tender and swollen. Tumefaction of thighs because of
menstruation. July 23. Delirium and insomnia with intense epigastric pains.
July 24, 9 p.m. Died.
Autopsy.--Performed 11 hours post mortem. Well nourished young woman,with well marked rigor morris and lividity. Body cyanotic and mottled. Pronounced general jaundice. Mouth and nostrils contained dark, partly dried
blood clots, and face was badly smeared with dried blood. Similar blood specks
were found on the hands and the genital regions, the skin of the thighs being
extensively excoriated owing to hemorrhages from the uterus. No petechi~e orsubcutaneous ecchymoses present. Lungs edematous. Pronounced hypostaticcongestion of the dependent parts. Several ecchymoses of pea size present.
Pleura free, containing moderate amount of deeply jaundiced clear fluid. On
section the hemorrhagic spots of the lungs were seen to extend many centimeters
into the parenchyma; some occult foci of similar ecchymoses in the interior.
Some old and some new loci of tuberculosis in the upper lobes. Pericardium apparently free from any changes; scanty icteric fluid in the cavity. Heart normal in size; in diastolic state, containing fluid blood in right heart. Few minuteecchymoses on the surface. Muscles pale, flabby, and brittle. Endocardiumand valves normal except for a few petechial hemorrhages on the papillarymuscles. Liver pale ocher-yellow in color, fatty, and streaked with congested
areas. Color on section uniformly yellow; parenchyma friable. Stomach distended with gas, containing blackish bloody mucous material; mucosa hyperemic,
especially a few minute hemorrhagic spots near the cardia. Intestines filled withdark tarry contents; mucosa hyperemic. Kidneys swollen and hyperemic, capsules non-adherent; on section numerous ecchymoses in the pelvis; increase of
the cortical layer and cloudy swelling. Deeply jaundiced. Suprarenal glands
hyperemic. Spleen and pancreas apparently normal. Ovaries congested anduterus hyperemic, containing extravasated blood in the cavity. Small amountof deeply yellowish urine in bladder.
Animal Inoculation.--The blood, drawn on the 5th day of disease, was used in
two different ways. One portion was immediately inoculated into the per i toneal cavity of two guinea pigs, and the other was put into a mixture of Ringer
solution 3 parts and human serum 1 part with 0.3 per cent neutral agar, then well
mixed and covered with a layer of paraffin oil. This semisolid mixture wasplaced in a thermostat at room temperature for 3 days (20-25°C.) and then injected into two guinea pigs intraperitoneally. The amounts of the original
blood injected into each guinea pig were from 2 to 3 cc. The protocols follow.

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T•xT-FIo. 1, a to f. Cases of yellow fever in which positive transmissions to guinea pigs were obtained.
(a) Case 1. Age 17 years. (b) Guinea Pig 129 A. Strain, Case 1. (c) Case 2. Age 23 years. (d) Case
3. Age 20 years. (e) Guinea Pig 353 C. Strain, Case 3. ~ ) Case 4. Age 15 years.

HIDEYO NOGUCHI

569

Guinea Pig 50.--July 19, 1918. Inoculated with 3 cc. of the fresh blood intraperitoneally. The temperature rose to 39.6°C. on the 6th day, dropped to38.5 ° on the 7th, again rose to 39.8 ° on the 8th, was 40 ° on the 9th, and 39.7 °on the 10th day. The animal became less active, and on the l l t h day it had atemperature of "39.1°, which continued for 3 days and then returned to normal(38-38.5°). On the l l t h day the scleras appeared icteric, and the capillaries of
the conjunctiva much suffused. The animal recovered completely in 15 days.
On a subsequent injection (Aug. 10, 1918) with material taken from other guinea
pigs inoculated with the enriched blood of the same patient this animal failed
entirely to react.
Guinea Pig 51.--July 19, 1918. Inoculated intraperitoneally with 2 cc. of thesame lot of blood. The temperature was 39.9°C. on the 9th day, 40.5 ° on the 10th,and 39.2 ° on the l l t h , returning gradually to normal (38.5-39°). There was asuspicion of jaundice on the scleras on the 12th day which disappeared in a few
days. This animal remained well and was subsequently (Aug. 10, 1918) tested
against material taken from this case. I t failed to react.
Guinea Pig 80.--July 22, 1918. 4 cc. of the mixture of the citrate blood andthe culture medium, which had been incubated for 3 days, were injected intraperitoneally into the guinea pig. The temperature was 40°C. on the 5th day,
40.1 ° on the 6th, and 38 ° on the 7th, when the animal became distinctly jaundiced.
Death occurred the same day (July 29).
Autopsy.--Marked general jaundice. No definite subcutaneous ecchymoses.
Dried blood specks around the nostrils. Mucous membranes very yellow. Lungs
congested; showed few disseminated ecchymotic spots which on section wereseen to extend deeper into the parenchyma. The size varied from a minutepoint to that of a split pea, and the color from bright red to dark bluish red.
The contour of these areas was usually sharply demarcated from ,the normal
tissue. Lungs edematous and the lower lobes highly hypostatic. Pleurae notaffected. Right heart appeared dilated and contained semicoagulated cyanotic
blood. A few punctiform hemorrhages on the anterior surface. Muscles friable.
Li~cer yellowish brown in color and mottled with areas of congestion. The markings of the lobules were prominent. Consistency firmer and less succulent than
the normal. Stomach distended with undigested food partly tinted with thedark blood which escaped from the hemorrhagic spots on the highly congested
mucosa, especially marked near the cardia. Intestines highly hyperemic andshowed numerous spots of hemorrhage along the mucosa. Contents bloody.
Kidneys intensely hyperemic and more or less swollen, showing a few punctiform hemorrhages in the cortex. On section the pelvis was filled with blood andthere were some ecchymoses. The cortex was broad and red, and the medulla
succulent and cloudy. Adrenals hyperemic and swollen. Spleen, pancreas,
bladder, and testes apparently unaffected. Along the abdominal wall themuscles were congested and showed some disseminated ecchymoses.
Microscopic Examination.--The blood was apparently free from any microorganism when examined under the dark-field microscope. Emulsions were570

ETIOLOGY OF YELLOW

FEVER.

II

prepared from the liver, kidney, and adrenals for examination by dark-field andalso for further passage. A very few actively motile organisms belonging to thegenus Leptospira were demonstrated in the liver and kidney emulsions but notin the adrenal.
Passage.--Two guinea pigs were immediately inoculated wiih a mixture of
the emulsions of the liver and kidney from Guinea Pig 80 and a further generation of this strain was obtained in the two following guinea pigs.
Guinea Pig 80 A.--July 29, 1918. The organ emulsion (2 cc.) was inoculated
intraperitoneally. The animal showed a temperature of 41.3°C. on the 4th day,
40.6 ° on the 5th, and 38.7 ° on the 6th, when the color of the skin and mucosa
became intensely icteric. Death occurred at 3 p.m. on Aug. 3 while the animal
was being placed in a mosquito cage with the stegomyias.
Autopsy.--The general appearance was the same as that of Guinea Pig 80.
The urine contained bile pigment, albumin, and abundant casts. The leptospira could not be demonstrated either in the blood, liver, or kidney.
Guinea Pig 80 B.--July 29, 1918. The emulsion was applied to the scarified surface of the skin. The highest temperature (morning) was 39.9°C. on the
7th day. I t went down to 38.8 ° on the 9th day, when the animal became very
yellow. The animal died on the 10th day (Aug. 7).
Autopsy.--About the same as in the foregoing experiment except for a higher
degree of degeneration of the liver, which was ocher-yellow, exactly as in somehuman cases. No leptospiras were demonstrated by the dark-field microscope,
but a further successful transfer was made.
Guinea Pig 81.--July 22, 1918. 5 cc. of the enriched blood of Patient A.
were injected intraperitoneally. The temperature was 40.3°C. on the 6th day,40~ on the 7th day, 39.9 ° on the 8th, and 38.7 ° on the 9th day, when jaundicebecame noticeable. The animal was found dead on July 31.
Autopsy.--Lesions similar to those already described in Guinea Pig 80. The
stomach was filled with a semifluid blackish gray matter (digested blood). Darkfield examination of the blood, liver, and kidney failed to demonstrate any organism. Transfers were made to six guinea pigs. The protocols are given
below.
Passage.--July 31, 1918. Six guinea pigs were inoculated with the emulsionsof the kidney and liver, intraperitoneally and also percutaneously after depilation.
Guinea Pig 124.--July 31, 1918. Kidney emulsion (1 cc.) intraperitoneally.
The animal had a temperature of 40.1°C. on the 4th, 40.5 ° on the 5th, 39.6 ° on
the 6th, and 39.9 ° on the 7th day. I t continued to show high temperature for
14 days, but jaundice was not present at any time. I t was discarded after 30days (Aug. 30).
Guinea Pig 125.--The same material was used as in the foregoing experiment.
The animal had a temperature of 40°C. on the 4th and 40.1 ° on the 5th day.
After that it was 39.5 ° or less until the l l t h day, when the animal became quite
icteric. It collapsed while being used for infecting stegomyias in a cage (Aug. 10,

HIDE¥O NOGUCm

571

4 p.m.), and was killed for examination of blood, liver, and kidney. Considerable
numbers of leptospiras were found in the blood and organs. Further transfers
were made with success. A culture was obtained from the heart's blood, but
within a week a fungus contamination destroyed it.
Passage. Guinea Pig 126.--July 31, 1918. The liver emulsion (1 cc.) of
Guinea Pig 81 was inoculated into the peritoneal cavity. The temperature went
up to 40°C. on the 5th, 6th, and 9th days; the animal showed definite jaundiceon the 10th day and died on the llth. The chief symptoms were al! very
marked.
G~inea Pig 127.--This was a duplicate of the foregoing experiment. The
animal died of a secondary infection within 3 days.
Guinea Pig 128.--Percutaneous inoculation with a mixture of the liver andkidney emulsions of Guinea Pig 81. The animal had a temperature of 40°C.
during the 10th, llth, and 12th days, but it had normally registered as high as
39.5°. There was a trace of jaundice on the 13th day, which soon disappeared.
The animal remained well for 31 days and was discarded as negative.
Guinea Pig 129.--This was a duplicate of the foregoing experiment. Temperature 40.3°C. on the 7th and 40.1 ° on the 8th day, when a suspicion of jaundice appeared. I t was killed on the 8th day in order to ascertain the lesions andobtain material for culture and transfers.
Autopsy.--Jaundice too slight to be definitely recognized. The lungs, how.
ever, showed the beginning of ecchymotic spots, and the liver and kidneys werehighly congested. Dark-field examination failed to reveal any organism. Transfers were made to two guinea pigs on the same day (Aug. 7). One of them diedwith mild but typical lesions, while the other completely recovered after having
shown a temperature of 40° and 40.4°C. on the 7th and 8th days.
Text-fig. 1, b is the chart of Guinea Pig 129 A, which succumbed to the intraperitoneal inoculation of 0.5 cc. of a culture of this strain (Aug. 27-Sept. 5, 1918),
with typical symptoms and lesions. The leptospira was demonstrated in theliver.
T h e foregoing experiments are of great interest as t h e y indicate t h a t
the microorganism which was responsible for the disease and subseq u e n t d e a t h of this p a t i e n t was a p p a r e n t l y successfully t r a n s m i t t e dto the guinea pig. T h e failure of the unmodified blood to reproduce the disease in guinea pigs as contrasted with successful transmission b y m e a n s of the same blood after a t e m p o r a r y enriching of
the virus in vitro m a y be explained b y the fact (as subsequent experim e n t s concerning the properties of the serum of yellow fever p a t i e n t s
and the properties of the organism indicate) t h a t the inoculation of
the fresh blood carried with it a certain a m o u n t of antibodies antagonistic to the d e v e l o p m e n t in the b o d y of the guinea pig, while this572

ETIOLOGYOF

YELLOW FEVEI~.

II

property is certain to be considerably modified when incubated in theculture medium employed. Moreover, as the organism is capable ofmultiplying outside the body at room temperature there is temporary enrichment of the organisms before inoculation into the guineapig.
I t is also shown in this experiment that the microorganism isvariable in virulence, being able to produce a fatal infection in some,an abortive infection in others, and in still others no infection at all.
The organism is capable of entering the body of the guinea pig insome instances by the smearing of the infective material. That there
is an abortive form of the infection is shown by the immunity that itconfers upon the guinea pigs which survived such an infection. The
infective agent may be passed from one guinea pig to another bytimely transmission.
A leptospira has been found to be associated with this case, but
its demonstration by means of the dark-field microscope is not always
successful; it may or may not appear in a subsequent passage. That
this organism, notwithstanding the difficulty of demonstrating it, isetiologically related to the disease known as yellow fever in Guayaquil
is made highly probable by subsequent experiments.
Fig. 1 shows the organism in the blood of a guinea pig inoculatedwith the culture of this strain.
The second positive transmission was obtained in the case ofPatient P.
Case 2 (Text-Fig. 1, c).--C. P., male, age 23 years; tailor, robust, a native of
Cuenca (highland), Had been in Guayaqull a month.
Onset July 20, 1918. Felt fever without chills; severe headache, photophobia, muscular pains throughout body, cramps in legs. No nausea. July 23.
Admitted to hospital. Marked injection of the conjunctiva; slight jaundice;much albumin in the urine. Liver small but tender (Dr. Elliott). Temperature
38.9°C. Blood was drawn from the median basilic vein and injected into twoguinea pigs (see protocols). July 24. Epistaxis in the morning. July 25.
Nauseated easily; diarrhea. Little urine since yesterday. July 26. Jaundiceadvanced. Albuminuria + + + . July 31. Improving. Temperature normal.
Aug. 2. Trace of jaundice. No albumin in the urine. Aug. 3. Discharged.
Animal Inoculation. Guinea Pig 88.--July 23, 1918. Intraperitoneal injection of 4 cc. of the blood of Patient P. (4th dayof disease). Temperaturerose to
39°C. on the 4th and 40° on the 5th day. The animal was killed on the 6thday
for further passages into two guinea pigs.

I-IIDEYO NOGUCHI

573

Autopsy.--Lungs showed a few hemorrhagic spots. Liver congested and somewhat yellowish (fatty). Kidneys congested and cloudy, showing swelling on section. Gastrointestinal system hyperemic but showing no hemorrhages. Spleennormal.
First Passage. Guinea Pig 88 A . - - J u l y 28, 1918. Received 1 cc. of blood ofGuinea Pig 88 intraperitoneally. The temperature rose to 39.9°C. on the 7thday; killed on the 10th day for examination and transfer.
Autopsy.--Slight icterus. The lungs were spotted with a few ecchymoses.
Liver congested and perhaps fatty. Kidneys congested and showed cloudy
swelling on section. Intestine hyperemic and showed scattered hemorrhagic foci.
The liver and kidneys were emulsified and used for inoculating two normal
guinea pigs (see second passage). Leptospiras not found.
Guinea Pig 88 B.--Duplicate of No. 88 A. This animal showed a slight riseof temperature in 4 days, but during observation for further evolution of thereaction it became normal and was discarded on Aug. 22.
Second Passage. Guinea Pig 89 A.--Aug. 6, 1918. 1 cc. of the mixed emulsion of the liver and kidney of Guinea Pig 88 A was given intraperitoneally.
The temperature went up to 40.9°C. on the 3rd, 39.6 ° on the 4th, and 40 ° on
the 5th day, but fell to 38.6 ° on the 6th day. Epistaxis and icterus. The animal
was found dead on the 7th day.
Autopsy.--Extremely jaundiced. Lungs hemorrhagic; liver yellowish; kidneys congested, with some ecchymoses. Stomach and intestines contained
blackish matter and numerous hemorrhagic areas were present on the mucosa.
Leptospiras were demonstrated in the liver and kidney but not in the blood.
Transfers to two normal guinea pigs were made, but this strain was lost because
of intercurrent infection.
Guinea Pig 89 B.--Duplicate of No. 89 A. This animal showed less fever thanusual in the 1st week but 40°C. on the 10th day. I t was kept under observation
until Aug. 22, but nothing further developed.
Guinea Pig 89.--This was one of the two guinea pigs inoculated with theblood of Patient P. on July 23. This animal showed a temperature of 39.3°C.
on the 4th, 39.9 ° on the 5th, 39.2 ° on the 6th, 38.9 ° on the 7th, 39.4 ° on the 8th,and 39 ° on the 9th day. I t was found dead on the 10th day.
Autopsy.--Congestion of the lungs, liver, and kidneys, but no icterus. No
transfer was made.
T h e t h i r d p o s i t i v e t r a n s m i s s i o n w a s o b t a i n e d w i t h t h e b l o o d of
P a t i e n t G.

Case 3 (Text-Fig. 1, d).--M. G., male, age 20 years; servant, robust, a nativeof Ambato (8,435 feet above sea level); had been in Guayaquil 2 months.
Onset Aug. 16, 1918, 10 a.m. General pain and aching; headache; fever without
rigor; no nausea, vomiting, or bleeding. Aug. 17. Admitted to hospital. Eyes
injected, face flushed; much prostrated. Pains in the head and limbs were574

ETIOLOGYO F YELLOW FEVER.

II

intense. Albuminuria slight; no casts. Temperature 39.9°C. Blood taken fortransmission experiments. Aug. 18. Albuminuria increasing; temperature 40.3 °.
Condition worse. Total leucocytes 9,200 (Dr. Elliott). Aug. 19. Albuminuria
increasing and numerous casts in the urine. Temperature still 40°C. Aug. 21.
Delirious; otherwise condition the same. Aug. 22. No urine obtained; gums
bleeding; jaundice definite; remission in fever. Aug. 23. Decided improvement;
temperature going down. Aug. 26. Beginning hematuria; increased icterus.
Aug. 29. Continued hematuria; great prostration. Aug. 30. Hematuria diminished; general improvement. Sept., 1. Continued improvement; cessationof hematuria. Sept. 2. Continued improvement; intense jaundice. Sept. 3.
Slight pain in bladder. Sept. 7. Discharged.
Animal Inoculation. Guinea Pig 351.--Aug. 17, 1918. 3 cc. of the blood ofPatient G. (2nd day of disease) were given intraperitoneally. The animal was
kept under observation until Sept. 7 but remained without any noticeable reaction except that its temperature rose to 40.1°C. on the 18th day. Discarded as
negative.
Guinea Pig 352.--Aug. 17, 1918. 5 cc. of the same specimen of blood were
given. Temperature 40°C. on the 7th day, 39.8 ° on the 8th, and 39.6 ° on the
9th, but later returned to normal (38.6 ° for this animal). Tested for immunity on
Sept. 7 against the A. strain and found to be resistant.
Guinea Pig 353.--Aug. 17, 1918. 6 cc. of the blood from Patient G. were
inoculated intraperitoneally. The temperature rose to 40°C. on the 7th and39.6 ° on the 8th day, but dropped to 38° on the 9th day. The animal was markedly icteric and was killed for examination of the blood and organs as well as
for transfer and cultivation of the virus.
Autopsy.--The lungs showed a moderate number of ecchymoses. Liver lightyellowish brown in color. Kidneys showed acute parenchymatous nephritis;no ecchymoses. Stomach contained a few hemorrhagic loci near the cardia withblood-stained contents. Intestines hyperemic but with few hemorrhages. Intense jaundice everywhere. Spleen normal in appearance. After a long searcha few leptospiras were discovered, but the number was so extremely small that
preliminary examination revealed none. Transfers were made successfully to
two normal guinea pigs, and cultures were also obtained. The organism wasdemonstrated in the blood of guinea pigs inoculated in later experiments with thisstrain (Fig. 2).
First Passage. Guinea Pig 353 A.--Aug. 26, 1918. Inoculated with 1 cc. of
the blood of Guinea Pig 353. Except for a slight rise in temperature nothing
happened during the period of observation (up to Sept. 10).
Guinea Pig 353 B.--Duplieate of the foregoing experiment. This animalcame down with typical symptoms and lesions on Sept. 5, 1918; that is, on the10th day after the transfer.
Second Passage. Guinea Pig 353 C.--Sept. 5, 1918. Inoculated with theemulsion of liver of Guinea Pig 353 B. The course of the infection was similar
to that of the positive instance, with the temperature as shown in Text-fig. 1, e.

HIDEYO NOGUCHI

575

T h e fourth positive transmission was o b t a i n e d with the blood of
P a t i e n t Co. Successful. transmission was also m a d e with the liveremulsion of this patient, as described below.

Case 4 (Text-Fig. 1, f).--J. C0., male, age 15 years; servant, native of Cuenca(highland). Had been in Guayaquil a month and a half.
Onset Sept. 2, 1918. Headache and pains in waist and lSgs; fever. Sept. 4.
Admitted to hospital. Face flushed; conjunctiva injected and slightly jaundiced;
gums reddish and swollen; tongue coated, with red tip and edge. Herpes labialis
present. Is No ~pigastric pain. Blood drawn for inoculation. Sept. 5. Passed
no urine, but appeared calm. Increased jaundice. In the afternoon became
excitable, anxious, c6mplained occasionally of dyspnea. Melena. Sept. 6.
Coma began previous night. Black vomit. Complete anuria. Death occurred
at 9 a.m.
Autopsy.--A partial autopsy within 1 hour after death. Liver highly degenerated and yellow. In the emulsion a few leptospiras were found. Kidney congested, cloudy swelling. No leptospiras.
Animal Inoculation. Guinea Pig 435.--Sept. 4, 1918. Intraperitoneal injection of 3 cc. of the blood of Patient Co. (3rd day of disease). The animal showeda temperature of 39°C. on the 8th, 40° on the 9th, 39.9" on the 10th, and 39 °on the l l t h day, but subsequently a normal temperature. It was discarded as
negative .after 25 days.
Guinea Pig 436.--Duplicate of the foregoing experiment. 5 cc. of the blood
injected. The temperature rose to 39.9°C. on the 9th and 40.1° on the 10thday, coming down to 38.2° on the 12th day. The animal showed distinct icterus
on the l l t h and 12th days and died during the night of the latter.
Autopsy.--General jaundice. Hemorrhages in the lungs and gastrointestinal
tract. Liver brownish yellow. Kidney highly congested; showed cloudy swelling on section. Other organs normal in appearance, Leptospira found under
the dark-field microscope and in stained slides (Fig. 3).
T h e fifth positive transmission was obtained with the emulsion of
the liver of the same patient, who died on the 5th day. T h e m a t e r i a l
was t a k e n from the b o d y within 1 hour, and the emulsion m a d e
with Ringer's solution was inoculated intraperitoneally into four
guinea pigs, the q u a n t i t y for each being 1 cc. A few distorted immobile leptospiras were d e m o n s t r a t e d in this emulsion u n d e r the
dark-field microscope.

Guinea Pigs 444 and 446.--Sept. 6, 1918. Received 1 cc. of the liver emulsion
from Patient Co. Died on the night of the following day.
t9 See Fig. 3, Paper I.

576

ETIOLOGYOF YELLOW

FEVER.

II

Guinea Pig 447.--Duplicate of the foregoing experiment. This animal nevershowed a reaction and died from an intercurrent infection after 22 days.
Guinea Pig 445.--Duplicate of the foregoing experiment. ' The animal had atemperature slightly above 39°C. for 6 days which rose to 39.9 ° on Sept. 13 andto 39.7 ° on the following day. Sept. 15. The animal was distinctly icteric andwas killed for examination and transfer.
Autopsy.--Typical lesions in the lungs, stomach, liver, and kidneys. The
blood, as well as the liver and kidney, showed a few leptospiras.
Passage.--From this animal the strain has been successfully passed throughmany generations. A culture also was obtained from a later passage.
T h e sixth positive tratlsmission was o b t a i n e d with the blood of
P a t i e n t Ch., d r a w n f r o m the m e d i a n basilic vein on the 5th d a y ofthe disease a n d 48 h o u r s p r e v i o u s to his death. I n this instance 2
cc. of the fresh blood were directly i n o c u l a t e d into the peritonealc a v i t y of each of the two g u i n e a pigs.

Case 5 (Text-Fig. 2, a).--E. Ch., male, age 30 years; well built man, a nativeof Guamote (highland). Had lived in Guayaquil for 8 months.
Onset Sept. 28, 1918. Sudden fever with chills, headache, pains in the limbs
and back. Admitted to hospital on Sept. 30. Still complained of pains in the
body and headache, with injection of the conjunctivm; tongue coated, with freeedge and tip; gums swollen. Albumin and casts in the urine, which was diminished in volume. Oct. 1. Scleras and skin somewhat yellowish. Oct. 2. All
symptoms aggravated. Oct. 3. Chest showed several pea-sized hemorrhagic
spots, slightly raised. Extensive ecchymotic areas were found on the skin where
the patient had applied mustard. 13 Jaundice marked; gums bleeding. Frequentemission of black vomit. The blood was transmitted to guinea pigs at 2.30 p.m.,
Oct. 4; condition becoming worse; anuria. Oct. 5, 3 p.m. Died.
Autopsy.--Performed 2 hours after death. Intense jaundice throughout theentire body. Skin over the chest showed numerous dark hemorrhagic spots.
Lungs much congested, showing many hemorrhagic spots. Liver bright yellowish in color, friable, and fatty. Kidneys highly congested, with cloudy swelling;some small ecchymotic loci in the pelvis. Capsules not adherent. Stomach
and intestines hyperemic with blackish bloody contents. Heart showed a few
ecchymoses in the pericardium and endocardium; muscles degenerated. Bladdercontracted with small amount of dark yellow urine. Spleen, pancreas, suprarenal glands, rand testes apparently unchanged.
Animal Inoculation. Guinea Pig 790 (Text-Fig. 2, d).--Oct. 3, 1918, 2.32 p.m.
Injected intraperitoneally with 3 cc. of blood from Patient Ch., on the 6th dayof disease. Temperature rose to 40.2°C. on the 6th and 39.8 ° on the 7th day
la See Fig. 1, Paper I.

577

HIDE¥O NOGUCHIFATALD A T E ~)CT. I
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Tv,xT-FIG. 2, a to & Direct transmissions from patient to guinea pig by meansof injection of the blood of Patient Ch., Case 5. (a) Case 5. Age 30 years.
(b) Guinea Pig 790 B. Strain, Case 5. (c) Guinea Pig 790 A. Strain, Case 5.
(d) Guinea Pig 790. Strain, Case 5.

578

ETIOLOGYOF

YELLOW FE%rER.

IIand came downto 37° on the 8th day, when the scleras and skin were intensely
jaundiced. The animal was rapidly weakening and was killed for examinationof the lesions and also for cultivation of material.
Autopsy.--Epistards; hemorrhagic lesions in the lungs and gastrointestinal
tract. Liver pale yellow and fatty, and kidneys' and suprarenal glands congested and swollen. Other organs apparently unchanged. In the blood andemulsions of the liver and kidneys the leptospiras were demonstrated. A pureculture was obtained from the blood of this animal.
Guinea Pig 791.--Duplicate of the foregoing experiment. The animal did not
become infected.
Passage.--Two guinea pigs were inoculated intraperitoneally on Oct. 11 with
1 cc. of blood from Guinea Pig 790, and both came down with typiqal symptoms
and lesions within 8 days (Text-fig. 2, b and c). The leptospiras were demonstrated in the blood and emulsions of the liver and kidneys. Pure cultures wereobtained from the blood of these guinea pigs.
T h e seventh positive transmission was obtained with a culture
derived directly from the blood of P a t i e n t A. Ce.

Case 6 (Text-Fig. 3, a).--A. Ce., female, age 18 years; seamstress, from a coast
town of Vinces. Had been in Guayaquil 12 days.
Onset with chills and fever on Oct. 16, 1918, followed by headache, pains in
the back, legs, and epigastric region. Vomited yellowish matter. Admitted on
Oct. 18. Slight jaundice; marked albuminuria; intense pain throughout thebody; nausea. Several petechial hemorrhages on the chest and arms. Oct. 19.
Jaundice more distinct; tongue coated, with free edge and tip; bilious vomit;
albuminuria; oliguria. Patient gravely ill. Blood culture made. Oct. 20. All
symptoms becoming worse. Black vomit and profuse bleeding from the gums.
Oct. 21. Comatose all day. Oct. 22, 8 a.m. Died. Autopsy not performed.
T h e culture tubes were examined on October 26, 1918, 7 days
after t h e y were set up, b y the dark-field microscope. Only one of
the six tubes showed the presence of active leptospiras in small
numbers. F r o m this twelve subcultures were made. T h e othertubes remained a p p a r e n t l y without growth. Inoculations into
guinea pigs were m a d e at three different laboratories, first in thel a b o r a t o r y of the Yellow F e v e r H o s p i t a l in G u a y a q u i l on October 27,
then in the l a b o r a t o r y of the Colon Hospital, P a n a m a Canal, on
N o v e m b e r 7, and finally in the l a b o r a t o r y of T h e Rockefeller Instit u t e on D e c e m b e r 11 and 19; t h a t is, 8, 17, 44, and 52 d a y s respectively after the time of m a k i n g the cultures. Subsequent subculturesttIDE¥O

579

NOCUC1TI

A.'TAL.

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TxxT-FIo. 3, a to d. Indirect transmission from patient to guinea pig bymeans of culture derived from the blood of Patient Ce., Case 6. (a) Case 6.
Age 18 years. (b) Guinea pig." Strain, Case 6. Second passage from culture.
(c) Guinea pig. Strain, Case 6. Culture 2. (d) Guinea pig. Strain, Case 6.
Culture 3.

580

ETIOLOGY O~F YELLOW FEVER.

IIof t h i s strain h a v e since b e e n o b t a i n e d (Figs. 5 a n d 6). T h e p r o tocols of some of these series of inoculations of g u i n e a pigs w i t h t h ecultures follow.

Animal Inoculatian (Second Series). Guinea Pig CeI2 (Text-Fig. 3, c).--Nov.
7, 1918. Inoculated intraperitoneally with 0.5 cc. of the culture (17 days old)of blood from Patient Ce. Temperature 39.8°C. on the 4th day, 40 ° on the 5th,
39° on the 6th, and 37.2 ° on the 7th. Suffusion of the conjunctivm, ears, andsoles. Slight jaundice appeared on the scleras, ears, and skin on the 7th day.
The urine contained bile pigment and 'albumin. The animal was killed on Nov.
13 for examination and culture.
Autopsy.--Moderate general jaundice. Many spots of hemorrhage in lungs;
liver congested and brownish dark red; kidneys swollen and cloudy; gastrointestinal mucosa hyperemie and somewhat hemorrhagic; spleen not altered.
Guinea Pig CeI3 (Text-Fig. 3, d).--The same as the foregoing experiment.
The animal showed symptoms and lesions almost identical with those justdescribed.
Animal Inoculation (Third Series). Guinea Pig 4, Second Passage from No. CeI2
(Text-Fig. 3, b).--Dec. 11, 1918. Inoculated intraperitoneally with 0.5 cc. of theliver emulsion of guinea pig infected with the first generation of culture of Ce.
strain (35 days old) which had never been passed through any animal. Temperature 39.5°C. on the 4th, 39.8 ° on the 5th, 40.6° on the 6th, 38.8 ° on the 7th,
41 ° on the 8th, 40° on the 9th, 38° on the 10th, and 37.6 ° on the llth day. The
animal became intensely yellow throughout the entire body and was killed for
culture and examination.
Autopsy.--All tissues were deeply jaundiced, and there were some minuteecchymoses in thesubcutaneous and muscular tissues. The lungs showed large
and small irregular hemorrhagic spots which on section were seen to extend into
the parenchyma. The color of the liver was strikingly yellow. Kidneys swollen
and on section cloudy; marking indistinct. Substance of kidney deeply jaundiced. Suprarenal glands congested. Mucosa of the gastrointestinal tract
hyperemic. Some hemorrhages, staining the contents blackish red. Spleen not
visibly affected. Only a few leptospiras found in the kidneys and none in the
blood or liver.
In addition to the animals given above five more guinea pigs were inoculated
in the same series. All showed symptoms and lesions similar to those justoutlined. Fig. 4 shows the organism in a stained blood preparation from oneof the infected guinea pigs.
T h e i n s t a n c e just described confirms the results o b t a i n e d in thefirst positive t r a n s m i s s i o n e x p e r i m e n t (Case A.); n a m e l y , t h a t t h eleptospira can be d i r e c t l y c u l t i v a t e d f r o m the blood of yellow fever
p a t i e n t s a n d t h e n i n o c u l a t e d i n t o g u i n e a pigs w i t h r e p r o d u c t i o n of

~rmxYo ~ o o v c m581

the symptoms and lesions which characterize the disease in man.
As we shall note again elsewhere, the virulence of this organism wasmaintained in cultivation for more than 5 weeks.
To sum up the results recorded in the preceding series of experiments, it is shown that there exists in certain cases of yellow fever
prevalent in Guayaquil a definite organism which is capable of beingtransmitted to the guinea pig and reproduces the symptom complex
characteristic of yellow fever. As other experiments showed, theorganism which has been observed and isolated in Guayaquil is a
leptospira (Figs. 1 to 6) closely resembling the Leptospira icterohcemorrhag#e of Inada and Ido, '° discovered by them in the infectious jaundice of temperate climates. That the present organism is closely
allied to but immunologically distinct from that species of the infectious
jaundice has since been established and the experiments bearing on
this point will be discussed in detail in subsequent papers. For thepresent I shall refer to the organism isolated from the yellow fever(typhus icteroides) cases as the yellow fever leptospira, or Leptospira.
icteroides (',Kr¢pos, jaundice, + ¢~n~, resembling, like), thus denoting
the source from which it was obtained.

Negative and Abortive Infections.
In the twenty-one instances to be recorded there was either a temporary febrile reaction in the guinea pigs after inoculation of theblood, with or without any suspicion of jaundice, or almost no reaction at all. In other words, the results are classed as indefinite or
negative. It is noteworthy, however, that a considerable proportion of the guinea pigs inoculated with the blood drawn during thefirst 5 days of the disease had a febrile reaction either on the 4th,
5th, 6th, or 7th day, and in some of these animals even a trace o fjaundice was noted or suspected for a day or two soon after the
fever. In none did the jaundice become definite, and the animals
subsequently returned to normal. Undoubtedly, by transferring thevirus to normal animals at this stage more strains might have been
secured in passage, but much time was lost in watching further development of the symptoms, and opportunities to obtain the strains
were lost. Some of these animals must have had a mild or abortive

582

ETIOLOGY OF YELLOW :FEVER.

IITABLE I.

Sex.

Course of disease.

Age.

Outcome of inoeulation.

yrS.

7
8

Male.

21
21

Severe; recovery.
"
"

2nd
2nd

2

"

9

"

19

Moderate; "

2nd

2

2nd

2

2nd

2

2

11

"

10

Severe; died on 7thday.
Extremely mild.

12
13
14

"
Male.

40
35
23

Moderate; recovery.
Mild;"
Severe;
"

2nd
3rd
3rd

"

25
25

Very mild;Moderate;

3rd
3rd

17

"

19

18

"

23

19

"

25

Severe; died on 6thday.
Severe; died on 7thday.
Severe; recovery.

20

"

20

21

"

28

22

"

16

10 Female. 26

15
16

23
24

"

"

"
"

16
20

25 Female. 21
26 Male. 21
27
"
32
27

"

32

"

"

"

"

Severe; died on 4thday.
Severe; died on 10thday.
Moderate; recovery.
Severe; died in 6(?) days.
Mild; recovery.
Severe;""died on 16th

3rd
4th
4th
4th
4th
5th
5th
ith(?)
6th
8th
4th

day.
i
Severe; died on 16th 15th
day.

2

2

Febrile reaction only.
""
in one, fever anc
trace of icterus in other; recov.
ery in both.
No reaction in one, fever andtrace of icterus in other~ recovery in both.
No reaction.
"in one, slight feverin other.
Febrile reaction in both.
"

"

"

"

2

"

No reaction in one, fever inother.
2 Fever in both.
2"""
and suspicion ot
icterus; both recovered.
1 Fever and suspected trace oficterus; recovered.
1 Fever and suspicious yellow tintin scleras; recovered.
2 Febrile reaction in both and suspicion of icterus in one.
2 Fever in both and perhaps a traceof icterus.
3 All had a febrile reaction but noicterus.
2 Both had fever and suspiciously
yellow scleras.
1 Febrile reaction.
2 Both had fever. Suspicion oficterus in one.
2 I Fever in one, no reaction in other.
2 No reaction in either.
2

"

"

"

"

2

"

"

"

"

HIDEYO NOGUCHI

583

form of the infection, as they subsequently proved to be refractory
to a virulent virus when tested after a period of about 25 days fromthe time of the inoculation of the yellow fever blood. For the sakeof completeness the protocols of the negative transmission experiments are recorded in Table I.
S U M ~ A R Y.

By injecting into guinea pigs the blood of yellow fever cases occurring in Guayaquil a group of symptoms and lesions closely resembling
those observed in human yellow fever were induced in a limited
number of instances. Of 74 guinea pigs inoculated with specimens ofblood from 27 cases of yellow fever, 8, representing 6 cases, came downwith the symptoms; namely, a marked rise of temperature after aperiod of incubation averaging 3 to 6 days, with simultaneous suffusion of the capillaries, particularly of the conjunctivae and soles,
then preliminary hyperleucocytosis followed by progressive leucopenia, the early appearance of albumin and casts in the urine, which
gradually diminishes in volume as the disease progresses. The fever
lasts only a few days, rapidly dropping first to the normal and thenusually to subnormal. At this period jaundice manifests itself invarying degrees of intensity, first in the scleras, then in the skin andthe urine. Hemorrhages from the nasal or gingival mucosa or anus
have been observed to occur during this period. Autopsies reveal
deep jaundice throughout the entire tissue. The liver is fatty andyellow, the kidney hyperemic, and often swollen and hemorrhagic.
Hemorrhagic spots were almost always found in the lungs and gastrointestinal rnucosa. Guinea pigs are usually rather sensitive tothe infection, though many appeared to be somewhat resistant andsome even refractory.
T h e injection of the yellow fever blood into ringtail monkeys,
rabbits, cats, guatusas, weasels, and sloths among the mammalians,and pigeons, ground-doves, bluebirds, mantas, blackbirds, parrakeets, reedbirds, blancos, and toucans among the birds, gave negative results.
In the blood, liver, and kidneys of the guinea pigs experimentally
infected with the blood of yellow fever patients a minute organism

58~

ETIOLOGY OF YELLOW F]BY'EI~.

II

was demonstrated which closely resembles in morphology the causative agent of infectious jaundice (Leptospira icterohcemorrhagice).
The leptospira transmitted from yellow fever cases to guinea pigs
was found to induce similar symptoms and lesions upon further
passage into normal guinea pigs.
The leptospira obtained from cases of yellow fever has been given
the provisional name of Leptospira icteroides.
I wish to express my thanks to Dr. Le6n Becerra, General Director of the Department of Health of Ecuador, and his staff for theirsupport and cooperation in the execution of this work; and likewise
to Dr. Pareja of the GuayaquU Yellow Fever Hospital and to hislaboratory staff (Dr. Larrea), clinical staff (Dr. Davila and Dr.
Martinez), and nursing staff for their invaluable assistance.
To Dr. Herman B. Parker I am indebted for his courtesy inlending me a dark-field arc lamp during the period before m y own
arrived, when the dark-field work would otherwise have been impossible, and also in furnishing me on various occasions with clinicalmaterial.
I am indebted to Colonel McCormack, Chief Health Officer of theCanal Zone, to Major Teague and Captain McFarland of the Ancon
Hospital, and to Captain Bowen and Lieutenant Levy of the Colon
Hospital, for the facilities afforded me in making renewal of cultures
and transfers of strains t o new animals.
EXPLANATIONO F P L A T E 35.

FIG. 1. Leptospira icteroides in the blood of a guinea pig experimentally inoculated with culture of strain from Patient A. A., Case 1. Fixed in methyl
alcohol and stained with Wright's stain. The film was made on the 6th day ofillness. X 1,000.
FIG. 2. The same; strain from Patient M. G., Case 3. X 1,000.
FIG. 3. The same; strain from Patient Co., Case 4. × 1,000.
FIG. 4. The same; strain from Patient A. Ce., Case 6. × 1,000.
FIG. 5. Dark-field view of a culture 1'6 days old of Leptospira icteroides.
Strain from Patient A. Ce., Case 6. X 1,000.
FIG. 6. The same. × 1,000.

PLATE 35.

THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X X I X .

FIG. 1.

FIG. -9.

FIG. 3.

FIG. 4.

Fit;. ,5.

FIG. 6.
(Noguchi: Etiology of yellow fever. II.)