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Journal of Experimental Medicine

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ETIOLOGY OF YELLOW FEVER.
Xl. Sxl~U~ TREATMENT OF ANIMALS INFECTED WITH LEPTOSPIRAICTEROIDES.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller In, titute for Medical Research.)(Received for publication, November 18, 1919.)
The high potency attained by a polyvalent immune serum for
Leptospira icteroides derived from the horse, as revealed in previous
experiments on guinea pigs# indicated the possibility that such aserum might be advantageously employed in the treatment of patients
suffering from an infection with that organism. In order to ascertain
whether or not the serum can exert a beneficial influence upon thecourse of the infection, several series of experiments were planned inwhich the guinea pigs were first inoculated with multiple minimallethal doses of Leptospira icteroides culture and then treated with theimmune serum at varying intervals afterwards.
Emphasis has been laid upon the fact that guinea pigs vary considerably in their susceptibility to Leptospira icteroides, ~ and instances
have been cited 1in which some animals survived after the injection ofa large amount of culture, while some succumbed to smaller amounts.
Irregularities of this nature were to be anticipated in the present
series of experiments, but this source of error was eliminated as muchas possible by using the serum in several graduated doses on a corresponding number of animals for a number of days in succession. In
some of the earlier series tests were rendered unsatisfactory by theuse of a culture which, from unknown causes, failed to kill the control animals, notwithstanding the fact that the same culture had
been highly virulent w h e n tested a fortnight previously. In anotherseries the virulence of the culture employed was such that the control
1Noguchi, H., J. Exp. Med., 1920, xxxi, 135.
2 Noguchi, H., J. Exp. Med., 1919, xxix, 585.
159

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ETIOLOGY OF YELLOW F E V E R ,XIguinea pigs died within 5 or 6 days after the injection of the culture,and the time for treatment was comparatively brief.
The serum was injected intraperitoneally in amounts of 0.001,0.01, 0.1, and 1 cc. at intervals of 1, 24, 48, 72, and 96 hours, andeven 5, 6, and 7 days, if the animals still lived, after the inoculationof the culture. In the earlier experiments two injections a day ofeach dose were given (10 a.m. and 4 p.m.), but this practice was soonabandoned in favor of a single daily injection.
In determining the effect of the immune serum upon the infection,
the temperature and other characteristic clinical symptoms werenoted, and the extent of the lesions was ascertained by killing thesurviving animals at a later period, when they were regarded asconvalescent. The lesions chiefly considered, although not a wholly
reliable index of the severity of the infection, were the hemorrhagicloci in the lungs.
In a series of therapeutic experiments in which the control guinea
pigs died within 5 to 6 days, a culture of Strain 5 of Leptospira icteroides being employed, it was found that when injections of theimmune serum were begun within 24 hours from the time of theexperimental infection (intraperitoneal), no symptoms or lesions
developed; provided the animal had received more than 0.001 cc.
of the serum. If the treatment was begun 48 hours after infection,
it was necessary to inject more than 0.01 cc. in order to prevent theirdevelopment. At the end of 72 hours, when some of the guinea pigs
had begun to show a rise of temperature, more than 0.1 cc. of theserum was necessary to check the progress of the infection, althoughsome animals treated with 0.01 cc. also recovered. After 96 hours,
when most of the animals had a high temperature, and some had
begun to show a trace of jaundice, the injection of more than 0.01 cc.
had a marked influence. The temperature came down to 100-102°F.
by the following morning, and no jaundice afterwards appeared inthese animals, which recovered within a week. A few guinea pigs,
which were slightly icteric at the time of injection of the serum,
became more deeply jaundiced on the following day and remained
so for a few days, when they began to convalesce. No guinea pig
treated within 96 hours with more than 0.01 cc. of serum died.

Hm~.YO NOG~Cm161

At the end of 5 days most of the animals which had not been treatedwith the serum showed jaundice and a decline in temperature, signsof approaching death within 24 hours. A few animals died in theafternoon of that day. To several of these critically ill guinea pigs
0.I to 1 cc. of the serum was gi~cen twice, at I0 a.m. and at 4 p.m.,but none of them seemed to be influenced by the injection. Somedied on the same day, others on the following day, showing all thetypical symptoms. At the end of 6 days several animals whichstill remained alive without the serum were treated with two dosesof 1 cc., but none was saved from death on the same day.
I t was found from this series of experiments that the progress ofjaundice, after it had existed for 24 hours, could not be checked by
the injection of the immune serum.
All the surviving animals were later killed for examination of thelung lesions: The animals treated with a sufficient amount (0.001
cc. or more) within 24 hours showed no lesions, or at most only a few
hemorrhagic spots in the lungs. On the other hand, fairly numerous
old hemorrhagic foci were found in the lungs of those which had beentreated at the end of 48 hours, indicating an infection aborted throughthe action of the serum. The lung lesions were decidedly more diffuse
and numerous in the guinea pigs which received the serum at theend of 72 and 96 hours, although none succumbed to the infection.
In the animals which died following treatment with the serum later
than 5 days after infection the symptoms and lesions were typicalof the infection and indistinguishable from those in the control animals which died without receiving any serum.
In Table I is given another series of experiments carried out with
Strain 6 of Leptospira ideroides. This culture killed control animals
in doses of 0.001, 0.01, and 0.1 cc. within 13, 10, and 7 days respectively, although one guinea pig inoculated with 1 cc. of the culture
escaped death after a severe infection. The amount of culture
employed throughout the series was 0.5 cc, and was given intraperitoneally. The injections of serum were begun 1 hour after theinoculation and repeated daily for 7 days, in doses of 0.01, 0.1, and1 cc.
As the table shows, no infection, so far as external manifestations
are concerned, took place in any of the guinea pigs injected with the162

ETIOLOGY OF YELLOW ~ E V E R .

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164

ETIOLOGYOF YELLOWFEVEIL

XIN?

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166

ETIOLOGYOF

YELLOBT FEVER.

XI

serum within a period of 72 hours from the time of inoculation withthe culture. All remained well.
At the end of 96 hours most of the animals had a temperature of103-104.5°F. in the morning. The injections of the serum weremade in the afternoon. The temperature remained high after theinjection but dropped gradually the following day, and the animals
recovered rapidly.
At the end of 5 days most of the animals had had a high fever for
2 days. The serum was given in the afternoon. Two of the threeguinea pigs treated began to improve the next day, one had a hightemperature for a day longer, but all eventually returned to thenormal condition.
At the end of 6 days the animals had begun to show more or lessjaundice and had had fever for 3 days. One guinea pig which received
0.01 cc. of the serum died 9 days from the time of infection, while
those which received 0.1 and 1 cc. recovered.
Experiments were made at the end of 7 days, when the animals
had had fever for about 3 days, and a slight decline had begun in themorning. In some there had been jaundice for 48 hours. The serum
was injected in the afternoon. The temperature the following morning
was quite high in most animals, and in some it lasted for 2 to 3 days,
while in others it went down rapidly. Jaundice increased in intensityin some, but gradually disappeared within several days. All recovered
except one, which was near collapse when 1 cc. of serum was injected
and which died the next day.
Of the surviving guinea pigs, no lesions were found in the lungsof those treated with the serum within 1 hour; and in each group of
three guinea pigs treated 24, 48, 72, or even as late as 96 hours (during
the incubation period) there was one animal in which no lesion
was present. In others, irrespective of the amount of serum given,
there were a certain number of hemorrhagic spots in the lungs, evenwhen treated with the serum within 24 hours. In one instance there
was no lung lesion in a guinea pig treated with 0.01 cc. after 5 days.
These irregularities are explained by the existence of a considerable
variation among individual animals in their susceptibility to icteroides
infection.

Hm~.YO NOGUCHI167

On the whole, the results obtained in this series admit of only oneinterpretation; namely, that the immune serum, when injected during
the period of incubation, prevents further development of the infection, and that when used in the early stage of the disease it is capableof preventing a fatal termination of the infection. On the otherhand, when guinea pigs are inoculated with a highly virulent culture,
the injection of the serum at a period of the disease when jaundice
has existed for some time and the animal is nearing collapse seems
to have no benefit. Undoubtedly the virulence of the strain employed
for the experiments has considerable bearing upon the eificacy withwhich the immune serum may be used in the late stage of the disease.
The less virulent the culture, the greater are the chances of benefit
from the injection of the serum after the clinical symptoms have manifested themselves, as shown by the favorable results obtained in thelast series of experiments just recorded. The fact must be emphasized, however, that even with a less virulent strain the serum has nobeneficial effect when given to a guinea pig which is in a condition
bordering on collapse (fall of temperature, with intense jaundice).
SUMMARY.

The use of a polyvalent immune serum of high potency, in thetreatment of an experimental infection of guinea pigs with Leptospira icteroides was found to be of definite advantage in checking theprogress of the infection. When administered during the period of
incubation the serum was found capable of completely preventing thedevelopment of the disease, although on subsequent examination
hemorrhagic lesions of greater or less number and extent were foundin the lungs of the guinea pigs which survived. Moreover, the serum
modified the course of the disease and when used in the early stagesof infection prevented a fatal outcome. Employed at a later stage,
however, when jaundice and nephritis had been present for several
days and the animal was near collapse, the serum had no perceptible
beneficial effect. This was, of course, to be expected in view of theincidence of various pathological phases of this disease--nephritis,
hepatitis, and other toxic symptoms in succession. In man theclinical manifestations are more gradual and distinct than in the

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ETIOLOGY OF YELLOW FEVER.

XI

guinea pig, 3 yet the yellow fever patient whose temperature is subnormal, and who has reached the stage of hemorrhages from thegums, nose, stomach, and intestines, and of uremia and cholemia,
would seem to have little or no chance of deriving benefit from theuse of a specific immune serum. This latter assumption would probably hold irrespective of the relation which Leptospira icleroides
proves to have to the etiology of yellow fever.
s Noguchi, H., Y..Exp. Med., 1919, xxix, 5~7.