野口英世著

Journal of Experimental Medicine

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E X P E R I M E N T A L STUDIES ON YELLOW FEVER INNORTHERN PERU.
BY HIDEYO NOGUCHI, M.D., ANn I. J. KLIGLER, 1M.D.
(From tke Laboratories of The Rockefeller Institute for Medical Researck.)(Received for publication, August 7, 1920.)

Leptospira icteroides, first isolated from certain cases of yellow feverin Guayaquil, 1 and later from a Case of this disease in Merida, ~ hadassumed such significance in the study of the etiology of yellow fever
as to make further investigations advisable. Peru, which has had
many visitations of yellow fever, was again invaded in June, 1919,
in the province of Piura, the northernmost region, bordering on
Ecuador. From this invasion a small epidemic arose which had notentirely disappeared in May, 1920. During the outbreak the following towns were affected: Sechura, Morropon, Tambogrande,
Chulucanas, Piura (500 cases among the 10,000 inhabitants) in 1919,
and Payta (108 cases among 3,000 inhabitants) in 1920. The mortality was estimated to have been about 10 per cent, which is considerably lower than was the case with yellow fever in Guayaquil and
Merida, where it was about 50 per cent. An expedition to Peru
was therefore undertaken.8 The present report deals with the resultsof bacteriological studies at Payta, Pinra, and Morropon extending
over a period of 3 months, March, April, and May, 1920.
When one of us reached Peru (March 1, 1920) Payta was the only
town where the epidemic of yellow fever was still in progress; thelast case occurred there on April 16. The first experiments were
1Noguchi, H., J. Exp. Med., 1919, xxix, 547, 565, 585; xxx, 1, 9, '13, 87, 95,
401; 1920, xxxi, 135, 159.
2 Noguchi, H., and Kligler, I. J., Y. Exp. Med., 1920, xxxii, 601, 627.
s This expedition was undertaken under the auspices of the International
Health Board of The Rockefeller Foundation, and The Rockefeller Institute for
Medical Research. We wish to thank the federal and local authorities in Peru
for their courtesy and cooperation in this work.
239

240

YELLOW F E V E R I N NORTIx'ERN PERU

carried out in Payta. In April an epidemic was reported at an
inland town, Morropon, and a trip was made to that place to securematerial for further studies to be carried out in Piura, where better
laboratory facilities were available.

Studies in Payta.
Payta, a town of about 3,000 inhabitants, is the principal port innorthern Peru. I t consists of a cluster of bamboo huts on a stripof sandy shore. Rain is rare, and water is very scarce. The watersupply comes from a river about 7 kilometers from the town, but
the amount is hardly sufficient for ordinary dally needs.
A provisional laboratory was set up in Payta in a small bungalow
consisting of three rooms. One room served as a laboratory, anotheras an animal room, and the third as a sleeping room. The laboratory
supplies brought from The Rockefeller Institute had been resterilized
in the laboratory of the Municipal Institute of Hygiene in Lima, as
there were no facilities for steam or hot air sterilization at Payta.
The rabbit serum used in the culture media was brought from New
York and as a result of the long voyage in a tropical climate a precipitate had appeared in it. The guinea pigs, also brought from New
York, had suffered severely, and about two-thirds of them had succumbed within 2 weeks of their arrival in Payta. The feed for theguinea pigs was scarce, so that only the larger and hardier animals survived. This was unfortunate, as the larger animals are less suitablefor initial inoculation; however, a certain number of native guinea pigs
was procured. A very serious circumstance was the fact that, owing
to the lack of electric current, the dark-field microscope could not be
used. Moreover, the effort to obtain Giemsa preparations of theblood were unsuccessful because of the quality of the water. Since
most yellow fever patients were treated by their physicians at theirown homes it was not always easy to secure consent to obtain bloodfor inoculation, and it was practically impossible to obtain blood
twice from the same patient. Finally, the cultures of Leptospiraicteroides brought to Payta from Merlda did not survive the journey.
Under the adverse conditions and the lack of laboratory facilities,
the bacteriological work was confined to cultivation and animal

HIDF.¥O NOGUCIIIAND

I. J. KLIGLEIR

241

transmission with such samples of blood as could be obtained, theobject being to infect guinea pigs and to produce the characteristics y m p t o m s and lesions in these animals. I n all, nine cases of yellow
fever were studied. Guinea pigs b r o u g h t from N e w Y o r k were
inoculated from seven and native guinea pigs from two of the cases.

Case I (Fatal).--B. Onset Mar. 10, 1920. Mar. 12. Temperature 39°C.;
pulse 110; albuminuria. Mar. 14. Temperature 38.5°; pulse 88; albumin 1 gm.
per 1,000 cc. Mar. 16. Delirious; black vomit; anuria. Mar. 17. Died.
Mar. 12 (3rd day of illness). Blood taken and cultures made (nine tubes).
Mar. 16. 1 cc. of eitrated blood (kept in the ice box for 4 days) was inoculated
intraperitoneally into Guinea Pig 1 and 2 cc. into Guinea Pig 2. The culture
tubes, which had stood at room temperature for 4 days, appeared free from contamination, and three guinea pigs were inoculated with material from Tubes 1 to
4 and three with material from Tubes 5 to 9. Some of the culture material wasleft standing until Mar. 23, when it was inoculated into three guinea pigs.
The two guinea pigs inoculated with blood 4 days old showed no fever. A few
old hemorrhagic areas were found in the lungs of Guinea Pig 2 when it was killedon the 15th day. Of six guinea pigs inoculated with culture material 4 days old,three had fever on the 6th day and showed petechial lung hemorrhages when
killed on the 15th day. The three remaining animals either suffered from anintercurrent infection (pneumonia) or escaped any obvious infection.
Of the three guinea pigs inoculated with the 11 day culture material, .one
(Guinea Pig 43) showed petechial hemorrhages in the lungs when killed on the
15th day. The other two showed on autopsy no leptospira lesions but enlarged
spleen and pulmonary congestion, which were taken as evidence of secondary
infection.
T h e findings described show t h a t in no instance was a fatal infection
b y Leptospira icteroides induced, b u t t h e y raise the question whether
the characteristic h e m o r r h a g i c areas in the lungs in Guinea Pigs
2, 3, 5, 8, and 43 did not indicate a mild infection with this organism.

Case 2 (Moderate; Reco~ered).--C. Onset Mar. 13, 1920. Moderate case;course typical. Mar. 20. Recovered.
Mar. 14 (2nd ~lay of illness). Blood taken and used wholly for making cultures. Three guinea pigs were injected with a 3 day culture, three with a 4 day
culture, and three with a 9 day culture.
Of this series almost all, except Guinea Pig 14, which received a 3 day culture,
and No. 19, which received a 4 day culture, died of intercurrent infections (pneumonia, paratyphoid, cocci), while two remained well. Guinea Pig 14 showedon the 5th day a temperature of 39.7°C., and on the 6th 39.6°, while at
autopsy the lungs showed several hemorrhagic areas. Guinea Pig 19 showed on_

242

YELLOW FEVER IN NORTtIERN PERU

the 7th day a temperature of 39.5° and on the 8th and 9th days 39.8~. It was
killed on the 10th day for examination and transfers. Few small petechia~ in thelungs and minute points of hemorrhage in the right kidney. The blood and organ
emulsions from this animal were inoculated on Mar. 28 into two guinea pigs,
both of which soon returned to normal.
The experiments on Case 2 are suggestive and lead to the tentative
conclusion that in two at least of the nine guinea pigs inoculated with
culture materials prepared with blood drawn on the 2nd d a y a mild
infection with Leptospira icteroides was induced. I t is possible t h a t
Guinea Pig 19, if allowed to live longer, might have developed atypical form of the icteroides infection, as, when killed on the 10thday, definite lesions were present in the lungs and kidney. T h e
failure to transfer the infection from this animal into two others is
not conclusive, as in the early transfers a l a r g e r n u m b e r of guinea
pigs should be employed, because of the resistance to infection which
certain guinea pigs usually exhibit.
Case 3 (recovered) gave similar results with blood drawn on the 3rd day ofillness.
Two guinea pigs were inoculated with 2 cc. of blood from Case 4 (fatal; blooddrawn on 2nd day of illness) soon after it was drawn, and two with the sameamount of blood from Case 5 (recovered; blood drawn on 3rd day of illness).
Cultures made with blood from each of these patients were left at room temperature for 4, 7, and 10 days and then inoculated into eight guinea pigs (Mar. 19,
22, and 26). Some of the animals inoculated with material from Cases 4 and 5
had definite febrile reactions and showed at autopsy lung lesions suggestive of amild leptospira infection, but there was no fatal infection with typical jaundice.
With Case 6 (recovered; blood drawn on Mar. 19, 2nd day of disease) and Case
7 (recovered; blood drawn on Mar. 20, 3rd day of disease), the blood was drawn
into citrate serum agar mixed in equal parts, and 3 cc. of the mixture were inoculated into each of two guinea pigs. Cultures made in the usual way were allowed
to stand at room temperature for 3 to 7 days, and two sets of six guinea pigs were
inoculated with this material The results with Cases 6 and 7, both with bloodand cultures, were unsatisfactory. The majority of the guinea pigs showed irregular febrile reactions, and from these animals, owing to the scarcity of guinea
pigs, no transfers were made. Some of them, when killed later, were found tohave hemorrhagic areas in the lungs, some showed indications of secondary infection (enlarged spleen), while others showed no lesions. In no instance was therea typical fatal leptospira infection.
Two _more cases were studied before the epidemic in Payta subsided, Case 8(recovered; blood drawn on 3rd day of illness, Mar. 29) and Case 9 (recovered;

HIDEYO NOGUCHIA N D I. J. K L I G L E R

243

blood drawn on Apr. 1, 2nd day of illness). In view of the failure to secure a deftnite transmissionwith fatal outcomewith the larger guinea pigs still alive from the
lot brought from New York, we decided to test native guinea pigs. For thispurpose six native and two American guinea pigs, weighing600 gln., were inoculated with a 7 day culture of the blood of Case 8 and five native guinea pigs
with the 10 day culture from Case 9. None of the animals developed a typieal fatal leptospira infection, although some undoubtedly had a mild infection,
since lung lesions were found at autopsy and in two instances there was a suggestion of jaundice. It is interesting to note here that later experiments demonstrated that the native guinea pigs possess a greater resistance to the icteroides
infection than the domesticated variety brought from the United States.
It is obvious that in the transmission experiments just describedas having been carried out at Payta, no typical instance of fatal
infection with Leptospira icteroides was obtained, and in no instance
was the leptospira observed under the microscope. As the darkfield microscope was not available and no proper Giemsa staining
could be secured , the latter cilcumstance is without value.
On the other hand, certain positive results were obtained in inoculated guinea pigs which led to the belief that a mild form of Leptospiraicteroides infection had in some instances been induced; i.e., rise of
temperature after the period of incubation common in this infection(3 to 5 days) and at autopsy definite hemorrhagic areas in the lungs
and in one instance in both lungs and kidney, with occasionally asuggestion of jaundice. The failures to obtain more pronounced
results are not difficult to account for. As stated above, almost all
the guinea pigs of the most favorable age and weight shipped fromNew York succumbed en route or soon after arriving at Payta. Thoseremaining were so few in number that they were used sparingly; hence
fewer were injected with given samples of blood or cultures than would
ordinarily have been employed. The rabbit serum which is essential
to successful cultivation of the leptospira had undergone changes
with the formation of a precipitate, and the reaction became so alkaline
as to prevent a growth of Leptospira icteroides to any extent. And
yet a degree of success, which was confirmed by subsequent results,
was, we believe, achieved.

244

n2~LLOW F E V E R IN NORTI-IERN P E R UStudies in Piura.
The epidemic having subsided in Payta, the laboratory was removed to Piura, where sterilizing facilities and adequate water wereavailable. A laboratory was set up, through the cooperation of thegovernment, in the Belan Hospital. A detached building was used
for animal quarters and feed was also more plentiful.
By the time the small laboratory had been started cases of yellow
fever were reported in Morropon, a small town of 2,000 inhabitantsin the foothills of the Andes. The distance from Piura was about
65 miles, a desert separating the two places. Arrangements weremade at once to investigate the cases? As the journey was madeon horseback it was obviously out of the question to transport experimental animals, etc., by this means through a tropical region; hence
it was decided to rely entirely on cultures. Fresh rabbit serum was
obtained from local rabbits, and in order to guard against adversechanges in the culture media the component parts, consisting ofserum, and 0.3 per cent semisolid agar, were carried separately.
The cult.ures were made b y drawing the blood from an arm veinof the patients directly into the tube of semisolid agar, the rabbit
serum then being added in a proportion of 1:5. The whole wasthoroughly mixed and covered with a layer of liquid paraffin, and thetubes were carefully capped with tin-foil and carried back to Piura.
On arrival at Morropon it was ascertained that cases of yellow fever
had been occurring for some time, and the epidemic was regarded as
declining; however, by mnklrtg house to house visits several cases
diagnosed as yellow fever b y Dr. Caballero were found on April 23.
Between April 24 and 27 cultures with the blood were made fromsix cases, one of which proved later not to have been yellow fever.
The remaining five cases pursued a clinical course which left no doubt
as to their yellow fever nature. Because of the illness of one memberof the party, the work at Morropon was suspended on April 28.
4 The journey on horseback from Piura to Morropon usually takes 1½ days.
Our party consisted of Dr. Enrico Caballero, the government expert stationedat Catacaos, who showed us every courtesy, Mr. John Mitchell, a sanitary engineer, and Dr. Kligler. The expedition started from Piura on April 21 andarrived at its destination on April 22.

ITIDEYO N O G U C S T I A N D

I. ~. K L I G L E R

245

The party returned with the cultures to Piura, reaching there on
M a y 3. At the same time the stock of guinea pigs had been renewed,
300 young, healthy animals having been brought from The Rockefeller Institute. Moreover, because of the lack of electric lighting
facilities which had made it impossible to use the dark-field microscope, a storage battery suited to that instrument had also beenbrought from New York.
Very few of the tubes showed contamination, the blood still appearing bright red in the upper zone of the media. The cultures were
inoculated into guinea pigs on M a y 6, Or 9 to 12 days after they
had been set up in Morropon. The inoculation procedure was
identical with that employed in Merida ~, the upper portions of several
selected tubes of culture from a case being pooled and the mixture
inoculated intraperitoneally into six young normal guinea pigs.
Dark-field examination of the culture tubes undertaken the nextday (May 7, 10 to 13 days after they were made) revealed the presenceof active leptospiras in the cultures from three of the five cases. They
were few in number and required careful search in some instances.
In some tubes no leptospira was detected. As the details of theexperiments show, the inoculation of cultures from four of the fivecases induced typical fatal infections in some animals, other animals
showing only a mild infection or escaping infection altogether.
Case 10 (Severe; Recovered).--C. M., male, age 16 years; born in Salitral; resident of Morropon. Onset Apr. 22, 1920, 7 p.m. Headache; backache; pains
in muscles; nausea; no vomiting. Apr. 23. Temperature 39.6°C.; pulse 106.
Apr. 24. Temperature 39.9°; pulse 100; albumin -~. Apr. 25. Temperature39°; pulse 90; albumin ~-~; nausea. Apr. 26. Temperature 37.4°; pulse 90;
albumin Jr -~; black vomit. Apr. 27. Temperature 37.6°; pulse 78; albumin ~c~;epigastric pain; epistaxis; urine increasing towards normal amount; jaundice.
Apr. 28. Temperature 37°; pulse 76; recovering.
Blood was taken on the 2nd day of illness at 11 a.m. Cultures examined after
12 days contained living leptospiras. Six guinea pigs (Nos. 13 to 18) were inoculated with material from Tubes 1 and 2 on May 6, with positive transmission inall.

C~i~a Pig 13.--Temperature 39.2° on the 5th day. Died on the 6th day.
Au~psy.--Epista~s; subcutaneous petechi~; marked hemorrhages in lungs
and gastric mucosa; jaundice slight.
The emulsions of kidney and liver were inoculated into three guinea pigs (Nos.
39 (Chart 1), 40, and 41), all of which developed typical fatal infection, dying

246

YELLOW F E V E R

IN N O R T H E R - ~ P E R U

7, 14, and 15 days after inoculation. The leptospiras were found and successful
subcultures made.
Guinea Pig 14.--Killed, when moribund, to obtain infective material fortherapeutic experiments to be described in the following paper. 5
Autopsy.--Numerous petechi~e in lungs; hemorrhage and blood in stomach;liver slightly degenerated; kidneys congested; spleen normal.
Guinea Pig 15.--Killed for transfer 4 days after inoculation, at first rise of
temperature to 39.4°C.
Autopsy.--No lesions were noted, but all three of the guinea pigs (Nos. 36,
37, and 38) inoculated with blood and emulsions of liver and kidneys succumbedwith typical infection.
C~inea Pigs 16 and/&--Developed characteristic infection. When jaundiceappeared they were utilized for testing the curative effect of the anti-ictero~tes
immune serum brought from The Rockefeller Institute, as will be described in thefollowing paper, sWith this case leptospiras were found in the initial culture, withwhich a typical infection was induced in guinea pigs, a n d f u r t h e rtransfer froth animal to animal was accomplished. P u r e cultures ofthe leptospira were in t u r n recovered f r o m the infected guinea pigs.

Case I1 (Mil~; Reco~ered).----O. V., male, age I8 years; native of Morropon.
Onset in afternoon of Apr. 21, 1920. Chills; headache; backache; fever. First
seen morning of Apr. 24. Temperature dO°C.; pulse 100; albumin +. Apr. 25.
Temperature 38=5°; pulse 70; albumin + + ; nausea, but no vomiting. Apr. 26.
Temperature 38.5°; pulse 66; albumin + + . Apr. 27. Temperature 37.2°;
pulse 58; albumin + ; abundaflt urine. Apr. 29. Temperature36.6°; pulse 50;
mild jaundice; recovering.
Apr. 24 (3rd day of illness). Blood was drawn. The dark-field examinationof cultures 13 days after they were made failed to reveal any leptospira, owing toaccidental contamination of the tubes while they were being handled the previous
day for animal inoculation.
May 6. Six guinea pigs (Nos. 25 to 30) inoculated with culture, then 12 days
old. Three of these (Nos. 25, 26 (Chart 2), and 29) developed typical severe
infections, while the remaining three showed no perceptible symptoms. Whenkilled for examination, however, all showed some hemorrhagic areas in the lungs,
indications of a mild infection. I t is interesting to note the different results withthe same culture material, due to variations in individual susceptibility of theguinea pig to Leptospira ictero~tes. The symptoms and lesions in fatally infectedanimals were altogether typical and hence will not be described in detail except inunusual instances. Leptospiras were found in the organ emulsions and a culture
was obtained from the blood.
5 Noguchi, H., and Kligler, I. J., J. Exp. Med., 1921, xxxili, 253.

HIDEYO NOGUCHI AND I. J. KLIGLER

247

Transfer from one of these animals was made into three guinea pigs, all ofwhich succumbed in due time to typical fatal infection.
One of the guinea pigs (No. 25) was used, when near collapse, for testing theefficacy of the anti-icteroides serum. The animal recovered, having received
1 cc. of the serum.
Case 12 (Fatal).--P. C., male, age 28 years; native of mountainous region.
Onset, Apr. 23, 1920, typical. Apr. 25. Seen for the first time; temperature
39.4°C.; pulse 102; albumin + + ; epigastric pain; no vomit. Apr. 26. Temperature 34.8°; patient ha state of collapse; bleeding from nose and gums; black
vomit; jaundice. Apr. 27, 6 a.m. Died.
Blood was taken in the morning of the 3rd day of illness. Cultures contained
living leptospiras when examined on May 7 (12 days old).
May 6. Six guinea pigs (Nos. 1 to 6) were inoculated with culture material.
Of these, three (Nos. 2, 3, and 4) developed severe infections, one dying on the
7th, and one on the 8th day (Chart 3), and the third being killed for transfer on
the 6th day, when it was intensely icteric. Three guinea pigs inoculated withblood and liver and kidney emulsions from this animal died with typical Symptoms. Three of the six original guinea pigs showed no sign of infection (Nos.
1, 5, and 6), but examination after 12 days revealed hemorrhagic areas in thelungs and also, in one instance, in the suprarenal. Leptospiras were found in
the blood and organs in some of the guinea pigs, and cultures were obtained fromthe blood. The outstanding feature of this strain was the predominance ofjaundice which it produced in the animals.
Case 13 (Moderate; Recovered).--J. C., male, age 14 years; native of Morropon.
Onset Apr. 23, 1920. Apr. 26. Seen for the first time; epistaxis; black vomit;
melena; temperature 37°C.; pulse 80; albumin + +. Apr. 27. Temperature 36.6°;
pulse 66; albumin + + . Apr. 28. Temperature 37°; pulse 80; albumin + + .
Recovery.
Apr. 26 (4th day). Blood taken for cultures. Dark-field examination of cultures 11 days after they were made failed to reveal any leptospira. Six guinea
pigs inoculated with the 10 day culture material from this case also yielded
negative results.
Case 14 (Severe; Recovered).--F. N., female, age 25 years; native of Morropon.
Apr. 25, 1920, 11 a.m. Onset. Apr. 26. Temperature 38.5°C.; pulse 114;
albumin trace; headache; backache; muscular pain; face flushed; conjunctivae
congested. Apr. 27. Temperature in morning 39.2°; pulse 106; albumin +.
4 p.m. Temperature 39.5°; pulse 120; severe pain and weakness; nausea, but
no vomiting. At request of patient 20 cc. of anti-icteroides serum brought fromThe Rockefeller Institute were injected intravenously. 7 p.m. Temperature
38.9°; pulse 100; stronger; pains relieved. Apr. 28. Afternoon temperature 38.2°;
pulse 100; albumin + + ; nausea; bilious vomit. Apr. 29. Temperature 38.2°;
pulse 90; no nausea; albumin + + . Apr. 30. Temperature 37.9°; pulse 86;
mild pharyngitis. May 1. Temperature 36.8°; pulse 80; albumin + ; recovering.

248

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CHARTS 1 to 4. T e m p e r a t u r e c u r v e s of guinea pigs i n o c u l a t e d w i t h m a t e r i a lfrom yellow fever cases in Peru.

HIDEYONOGUCHIAND

I. ]. K L I G L E R

249

Apr. 27 (48 hours after onset). Bloodtaken in the morning. The cultures were
examined on May 8 when 11 days old, and living leptospiras were found in one
tube, apparently dead ones in another.
May 6. Six guinea pigs (Nos. 7 to 12) were inoculated with the 9 day culturematerial. A typical infectionwas inducedin three, while the other three remained
apparently well. One of the latter showed hemorrhagic areas in the lungs when
examined after 12 days. The other two had no macroscopic lesions, indicatingthat they were both completelyrefractory to this strain.
Transfer was made from one of the positive animals into three guinea pigs,
all of which died with typical infections. Leptospiras were demonstrated invarying number in the blood as well as in the emulsionsof liver and kidneys, andpure cultures were obtained from the blood of these animals. Chart 4 shows thetemperature curve of one of the guinea pigs (No. 45) infected with Strain 4.
In Case 14, as in Cases 10, 11, and 12, positive transmission t oguinea pigs was obtained by means of culture material. The initial
cultures usually contained riving leptospiras. In the blood and liveror kidneys of the infected animals the leptospiras were demonstrated,
and a pure culture of Leptospira icteroides was recovered from theblood.

Identification of the Morropon Strains.
Upon our return to The Rockefeller Institute, we proceeded with
the identification of the strains of leptospira isolated from yellow
fever cases in Morropon along the lines previously followed. The
Pfeiffer phenomenon was determined, as well as the effects of immune
serums upon the organism in vitro.
Rich cultures of leptospira strains (Nos. 1 and 2) were employedin these experiments. The serums used were monovalent immune
serums prepared in rabbits with Guayaquil Strain 1 of Leptostrlra
icteroides. For purposes of control American Strain 2 of Leptospira icteroh~raorrhagice was tested simultaneously. Moreover, a
polyvalent immune serum prepared in a horse with several Guayaquil
strains of Leptospira icteroides was also tested. The results obtained
are given in Table I.
As Table I shows, the leptospira strains from Morropon gave
positive Pfeiffer reactions with the immune serums prepared with
the Guayaqull strains, but negative reactions with the anti-icterohcemorrhagi¢e serum. Likewise, with respect to their behavior towards

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YELLOW F E V E R

IN NORTHERN

PERU

these serums in dtro an indubitable specificity for the anti-icteroides
serum is evident. The slight reaction with the anti-icterohcemorrhagice serum may be regarded as a group reaction among closely
allied species. It is concluded, therefore, that the leptospiras isolated
from Morropon and Guayaquil cases of yellow fever are of the same
species.
TABLE I.

Identification of the Morropon Strains.
Mode of test.

?feiffer (30rain.).

rn vitro; al-lowed tostand for18 hrs.

Anti-iaeroh~'m~rStrain Anti-iaeroldes serum Anti-icteroides serum rhagie~
without
serum (mono- Controls
immuneserum.
No. (monovalent,rabbit). (polyvalent,horse). valent,
rabbit).
1

Complete disin- Complete disin- For the most Very active
tegration
tegration
part active(negative).
(positive).
(positive).
(negative).
Almost comi Complete disin- For the most
Very active
plete disintetegration
part active;(negative).
gration (posi(positive).
few appeartive).
distorted(negative).
Complete agComplete agSlight aggluti- Very active; noagglutination,
glutination;
glutination
nation andsome motile
and immobil- few immobilleptospiras.
ization.
ized.
Complete agComplete agPartial aggluti- Active; no aggluglutination;
glutination;
tination.
nation andimmobilizasome motile
few motile
leptospiras.
tion.
leptospiras.

DISCUSSION AND SUMMARY.

Fourteen typical cases of yellow fever were studied in northern'
Peru during an epidemic occurring in 1920, nine in Payta in Marchand April, and five in Morropon and Piura in April and May. The
method of investigation was similar to that previously employed, but
as the laboratory facilities were very meager certain changes wererequired. Although in Payta the work was handicapped by the lackof electric light, the scarcity of water and animal food, the unsuitability of the guinea pigs for inoculation, and the changes in culture
media due to age, the results obtained under these adverse conditions
were by no means negative. While in no instance was there a typicalHIDEYO NOGUCHI AN'D I. J. KLIGLER

251

infection produced in animals, either by direct inoculation of bloodor with culture materials, yet certain guinea pigs in each series showedtemporary febrile reactions or definite hemorrhagic lesions of t h elungs indicative of a mild leptospira infection. Direct search for
Leptospira icteroides in the blood of patients or in cultl~re materials
was not made because the dark-field microscope could not be used.
Subsequently, at Piura, the laboratory facilities were vastly, improved, the use of the dark-field microscope was made possible bymeans of a storage battery, and a fresh stock of young healthy guinea
pigs was received from New York, and fresh rabbit serum obtained in
Piura. In the study of the materials obtained from five cases ofyellow fever in Morropon all these added facilities were taken advantage of, with the result that the outcome was positive and convincing. Cultures from the five cases were examined after 11, 12, and13 days, and in those from three cases living leptospiras were found.
By inoculation into suitable guinea pigs of culture material fromthese five cases, irrespective of whether or not leptospiras weredetected under the dark-field microscope, a typical Leptospira icteroides infection was produced from four of the five cases. In one ofthese no leptospira had been detected in the culture tubes. Thus
one case only yielded negative results, in that no leptospiras were
found under the dark-field microscope and the animal inoculation
was negative.
The leptospira was demonstrated in the blood or organ emulsionsof the infected guinea pigs, and further transmission of each strain
to other guinea pigs was obtained and pure cultures were secured.
A few points of practical significance appeared in the course of thepresent investigation. One is the importance of using fresh rabbit
serum for culture media. Old rabbit serum, whether in pure form
or incorporated with agar, etc., which had been kept for several months
in a tropical climate, proved to be unsatisfactory for obtaining a
growth of Leptospira icteroides. A second point of interest is thevariation in susceptibility of guinea pigs to infection with Leptospiraicteroides. In two of four series of positive animal inoculations with
the Morropon culture materials only one-half of the guinea pigs
inoculated with given materials developed typical symptoms. The
other half either suffered from a transient mild infection, as evidenced
by a few hemorrhagic foci in the lungs, or escaped infection altogether.

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Y~LLOWF E V E R IN N O R T H E I C N PEI~U

From these facts it is highly probable that the lung lesions andfebrile reactions observed in certain guinea pigs inoculated with the
Payta materials were due to a mild leptospira infection. In a comparative experiment the native guinea pigs procured in Payta were
found to be more resistant to the leptospira infection than thoserecently brought from New York. In fact, only a small portion of
the former succumbed to typical infection even when inoculated witha virulent strain of Leptospira icteroides obtained from the Morropon
epidemic.
In conclusion it may be stated that of fourteen cases of yellow feverstudied in Peru, a typical leptospira infection, together with thedemonstration of the organism in experimentally infected guinea
pigs, was obtained in four, while in the majority of instances indications of a mild, non-fatal leptospira infection were observed. I n a
few cases only were the results entirely negative.
The leptospira isolated from Morropon cases of yellow fever, which
is morphologically and culturally identical with the Guayaquil and
Merida strains of Leptospira icteroides, was also shown by immunity
test to be indistinguishable from the Guayaquil organism.