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Journal of Experimental Medicine

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ETIOLOGY OF YEI,LOW FEVER.
VII. DEMONSTRATION OF LEPTOSPIRA ICTEROIDES IN THE BLOOD,
TISSUES, AND URINE OF YELLOW
FEVER PATIENTS AND OF
ANIMALS
EXPERIMENTALLY
INFECTEDWITH
ORGANISM.

BY IIIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, May 16, 1919.)
The number of Leptospira icteroides found in the blood of yellow
fever patients was so small that a prolonged examination of blood
specimens was necessary to discover one organism, even when positive transmission had been obtained by injection of the blood into
guinea pigs. In most cases the organism could not be detected in theblood, perhaps owing to lack of time. To devote much time to
microscopic examination during the period while the work with yellow fever patients was being carried on was impossible and also inadvisable, since it was a matter which could be taken up at a later
date when more time was available. Hence, what will be reported
in this paper is not final but is the preliminary account of what wasaccomplished under the circumstances. The demonstration of theorganism in the blood and various organs of animals experimentally
infected with Leptospira icteroides, on the other hand, was much moresatisfactorily accomplished. The infection could be produced at thedesired time and the material obtained in sufficient quantity at any
stage of the disease.

Examination of Material from Yellow Fever Cases.
As already stated I a minute organism belonging t o the genusLeptosp#a has been found both in the blood and in the tissues ofvarious.organs, particularly the liver and kidneys, of guinea pigs
i Noguchi, H., Y. Exp. Med., 1919, xxlx, 565.
87

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ETIOLOGYO F YELLOW F E V E R .

VIIwhich contracted an experimental infection after being inoculatedwith the blood of yellow fever patients taken during the early stagesof the disease. In three instances (Cases 1, 4, and 6) the organism
was found in the cultures made directly from the blood of yellow feverpatients. A special effort was made, therefore, whenever a case ofyellow fever was admitted to the hospital or an autopsy was performed, to find the same organism in the blood and tissues, and if
possible also in the urine.
The dark-field microscope was used for examination of the freshmaterial. Films and sections were stained by Giemsa's or Wright'sstain, by Levadifi's or Noguchi's silver impregnation methods, asoccasion demanded. Fontana's silver impregnation method was applied several times without satisfactory results. Blood films wereusually fixed with methyl alcohol,2 while the impression films ofvarious tissues from autopsies were first fixed over osmic acid vapor
for 1 minute and then in absolute alcohol for 30 minutes. They werestained with Wright's stain for 30 minutes and then with Giemsa's
stain over night. Six to twelve were prepared from each specimen ofblood or tissue for examination.
Of twenty-seven cases of yellow fever in only three (Cases 3, 4,and 14) was the leptospira demonstrated in the blood under the darkfield microscope, and in each instance the number of the organisms
found was so small that once they had passed out of the field they
were not easily encountered again. The blood of two of these threepatients (Cases 3 and 4) also yielded positive transmission into
guinea pigs. 1 The organism was found in a few of the stained film
preparations, but these will have to be repeatedly examined. Some
specimens of yellow fever blood, however, were infectious for guinea
pigs even when in the fresh state no leptospira could be demonstrated
under the dark-field microscope (Cases 1, 2, 5, and 6). That there
must have been a very small number of the organisms in such specimens is shown by the fact that the blood and organs of the infectedanimals contained the organisms. A negative microscopic examination of the blood indicates either absence or scarcity of the organism.
Nor does the failure of the blood to reproduce a fatal experimental
For the purifying of the methyl alcohol for this purpose I am indebted toDr. Herman Edward Redenbaugh of the Commission.

HIDEYO NOGUCHI

89

infection in guinea pigs prove the absence of the organism in thespecimen, since variations in its pathogenicity for guinea pigs are
considerable (Case 14).
Examination of various organs under the dark-field microscope
has yielded so far only one positive finding, that in the liver ofPatient J. Co. (Case 4), who died on the 4th day of the disease. Theliver was excised from the body (a partial autopsy) a few hours after
death while the body was still quite warm. The kidney failed toshow any leptospira. In the stained specimens of the blood, liver,and kidney a small number of leptospiras was demonstrated. Boththe blood and the liver emulsion of this patient yielded a positivetransmission of the disease to guinea pigs. 1
A careful search for the organism was carried out with the 6Ires
made from the liver, kidney, lungs, adrenals, mesentery, and inguinal
glands from ten more cases, with so far a positive finding in the liverof one case (Case 5). Some of these slides were poorly stained andwill have to be repeatedly examined when more time is available.
In a later paper will be recorded the results of the examination ofsections of the organs from patients who died of yellow fever.
Specimens of urine from twenty-one cases of yellow fever wereexamined under the dark-field microscope, but no leptospira was encountered. The examinations were made during the height of theillness, which is usually the 5th or 6th day of the disease, as well as
during convalescence, towards the end of the 2nd week. This partof the investigation was much handicapped by the lack of a powerfulcentrifuge to concentrate the urine, and it will have to be repeatedunder more favorable conditions. As might be expected, Treponema
minutum 3 and Treponema calligyrum 4 were occasionally seen in thesamples of urine. Adoubtful result was obtained in a guinea pig inoculated with 10 cc. of the urine from a convalescent (Case 51) on
the 15th day of the disease, although no leptospira was found in thespecimen.
8 Noguchi, H., .1".Exp. Meal., 1918, xxvii, 667.
4 Noguchi, H., Y. Exp. Med., 1913, xvii, 89.

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ETIOLOGY OF Y E L L O W F E V E R .

VIIExamination of Material from Experimentally Infected Animals.
Experiments were undertaken to determine the approximate time
when the blood, liver, and kidney of guinea pigs infected with theyellow fever virus first contain enough organisms to produce the infection when transferred to a normal guinea pig, and when they cease
to be infective. These points are more of academic interest, as they
do not affect the present sanitary measures with regard to isolationof the patients before, during, and after the actual sickness.
When a minute quantity of the virus is introduced into the subcutaneous tissue of the guinea pig no local reaction follows. The
blood becomes infective as early as 48 hours after the inoculation insome instances, but more constantly so after 72 hours. The liverand kidney become infective simultaneously with the blood. The
amount of blood effective for infection is comparatively large, atleast 1 cc. being required during the earlier period. The demonstration of the organism in the blood is almost hopeless and has not beensuccessful until the 5th day after inoculation, when occasional specimens have been found after a long search. The organisms are more
readily found in the emulsions of the liver or kidney, sometimes more
numerously in one and sometimes in the other. After the onset of thedisease the number of organisms gradually increases for a time, bothin the blood and in the liver and kidney. Then the relation of theorganism to the blood on the one hand and to the liver and kidneyon the other seems to show a certain difference. The organisms continue to increase in the organs somewhat longer than in the blood. In
fact, the number of organisms in the blood becomes fewer as thedisease progresses, and only a few can be detected when the jaundiceand other symptoms have fully developed; thatis, a day or two before death. This has been the rule with the majority of the guinea
pigs in the present experiments, but there were exceptions in which
the organisms were quite numerous until death. Although they increase in the liver and kidney in greater number and for a longer
period, they have been found in most instances finally to disappear
also from these organs. The disappearance is sudden and usuallyoccurs about 24 hours before death, although there are instances inwhich they can be found in large numbers at death, sometimes

IIIDEYO NOGUClZI9t

more abundantly in one or t h e other organ. Although it is truethat when the blood contains the leptospira at the time of death theliver and kidney also contain it, yet the reverse is not always the case,the organism being found in the organs but not in the blood. A n
instance has not been met in which the leptospira was present in theblood but not in the liver or kidney or both.
The mechanism of the disappearance of the leptospira in so many
typical experimental guinea pigs during the later stage of the infection is difficult to explain, but it may be partly due to the forn~ation of certain still undetermined metabolic products brought about
by the disorganization of the liver or kidney or some other organs.
The leptospira is highly sensitive to the dissolving action of bile salts
in vitro, and it is not inconceivable that a predecessor or a derivativeof taurocholic, glycocholic, or cholic radicals in a certain stage mayexert a powerful destructive action upon it.
With regard to the question of the infectivity of the blood andorgans in the later stage of the infection, the statement may be madethat in all' instances in which the organism was seen, whether in theblood or in the organs, the material was always infective. On the'other hand, in a considerable number of ihstances in which thedark-field search for the organism failed, no infection could be induced in normal guinea pigs by inoculation of the material. It may
be supposed t h a t the organisms had completely disappeared from theb o d y in these cases. However, as was to have been expected, there
were many instances in which, notwithstanding an unsuccessful microscopic search for the organism, inoculation of the liver or kidney, orboth, but not the blood, gave rise to a typical infection in furtherpassage, with the reappearance of the leptospira in the new host.
In one instance the leptospira was microscopically detected on thethird passage. It may be concluded, then, that in experimental
infection in the guinea pig the specific organism survives longer insome of the infected hosts than is assumed to be the case in y e l l o w
fever in man.
SUMMARY.

Examinations of fresh blood from yellow fever patients by meansof the dark-field microscope, made in rnorc than twenty-seven cases,

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

VIIrevealed in three cases the presence of Leptospira icteroides. In noinstance was a large number of organisms found, a long search being
required before one was encountered. The injection of the blood
into guinea pigs from two of the three positive cases induced in theanimals a fatal infection, while the blood from the third positive casefailed to infect the guinea pigs fatally. Careful but by no means exhaustive dark-field searches for the leptospira with fresh specimens ofblood from the remaining cases of yellow fever ended without positive findings, although four of the specimens, when injected into
guinea pigs, caused a fatal leptospira infection.
Stained blood film preparations from the corresponding cases werealso examined, but the percentage showing the leptospira in the blood
was no greater than that found by examination in the fresh state withthe dark-field microscope. In fact, owing to the defective stains thatwere available at the time of the investigation a great many slides did
not take the proper coloration with Giemsa's or Wright's stain andcould not be relied upon.
Regarding the presence of Leptospira icteroides in various organs
b o t h dark-field and stained films were examined. In only one instance so far a few organisms were detected in the emulsion of liver
taken shortly after death from a case dying on the 4th day of yellow
fever. This part of the work will be reported later upon completion.
Examinations of the urine from different cases of yellow fever
were made both by dark-field microscope and by inoculation into
guinea pigs. The results were totally negative in thirteen cases, including many convalescents, but in one case one of the guinea pigs
inoculated with 10 cc. of the urine came down on the 15th day withsuggestive symptoms (suspicion of jaundice, and some hemorrhagicand parenchymatous lesions of the lungs and kidneys). This specimen showed no leptospira by dark-field examination.
In experimental infection of guinea pigs with Leptospira icteroide~
the blood became infective in many instances 48 hours after inoculation, and was always infective after 72 hours. The liver andkidney become infective simultaneously with the blood. Detectionof the organism b y means of the dark-field microscope has seldom
been accomplished before the 5th day. The organisms are mostabundant on the 6th to the 7th day, but become fewer or completely

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93

disappear before death. In the meanwhile the number of organisms
increases in the liver and kidney, from which they disappear as thejaundice and other symptoms become aggravated. When deathoccurs these organs seem to have lost most of the leptospira, andpositive transfer b y means of them is less certain. At the laterstage of the disease the blood is often free from the organisms andceases to be infective. Positive transmission with blood obtained
from moribund animals is not impossible, however, even when no
leptospira can be detected under the dark-field microscope.