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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
X l I . INFLUENCE OF MALARIAL INFECTION (PLASMODIUM INto?),SPLENECTOMY, O1~ BOTH, UPON EXPERIMENTALCARRION~S DISEASE IN MONKEYS.
BY HIDEYO NOGUCHI, M.D.

(Front #he Laboratories of The Rockefdla" Ins~ilute for Medical R,se~rch.)(Received for publication, January 6, 1928.)

Mixed infections with Bartonella bacitliformis and plasmodia are
not uncommon in regions where both malaria and Carrion's disease(verruga peruana, Oroya fever) are endemic. Both parasites induce
swelling of the spleen, and both invade the red blood cells. It isconceivable that the preexistence of malaria might have an unfavorable influence upon a subsequent infection with Bart~nella bacilliformis,
as a result of impairment of the defensive powers of spleen and bloodby the plasmodia, or that the introduction of plasmodia during thecourse of a dormant or partially controlled verruga infection might
result in renewed invasion of the blood and lymphatic system byBartonella bacilliformis. The abrupt development of malignant Oroya
fever in the course of apparently benign verruga would be explicable on
this latter hypothesis.
The opportunity to obtain definite experimental data on the effectof malaria infection on the course of experimental verruga presented
itself when malaria parasites were found in the blood of a monkey(Macacus cynomdgus) which had been splenectomized in connection
with an experiment to determine what influence the operation would
have upon the susceptibility of monkeys to Bartonella baciUiformls.
Protocol 1.--Macacus cynomdgus l-M, splenectomized on Dec. 1, 1926, to serveas control for M. cynomolgos 2-S, which was splenectomized the same day andinoculated with Bartonella bacilliformis. Examination of the blood of Monkey
1-M was made daily in order that the appearance of any Bartondla-like intracor821

822

ETIOLOGY OF OROYA

FEVER.

Xllpuscular elements (such as appear in splenectomized rats t) might be detected.
These elements were not found, but the examinations revealed plasmodia~ on
Dec. 6, 9, 10, 11, and 16, 1926, and the parasites were still present at the time ofdeath on Mar. 10, 1927. No fever was detected ill this animal at ally time. Themonkey had shown no signs of illness during several months of captivity previous
to the operation. It would appear that it was a chronic malarial parasite carrier,a condition already established for man. Death may have been due to aggravation Of the malarial infection through splenectomy.

Preexisting or Concomitant Malarial Infection and Verruga.
A Macacus rhesus intravenously inoculated with the blood of
Macacus cynomolgus 1-M on D e c e m b e r 10 soon developed fever, a n d
the malarial parasites were found in the blood. One m o n t h laterv e r r u g a materi~l was i n t r a d e r m a l l y inoculated. I n due course thecutaneous lesions a p p e a r e d a n d a t t a i n e d m o d e r a t e size. Blood t a k e non F e b r u a r y 3, 1927, yielded cultures of Bartonella bacilliformis, b u t
no severe f o r m of blood invasion and a n e m i a developed. T h e a n i m a lrecovered f r o m b o t h infections within a few weeks.
Protocol 2.--Macacus rhesus 2-M, inoculated intravenously with 0.5 cc. of theMood of Monkey 1-M on the day when plasmodia were first noticed (Dec. 10,1926). The febrile reaction began on Dec. 15, that is, 5 days after inoculation, andcontinued daffy for 9 successive days, being high (105-106.2°F.) for 5 days andmoderate (104.2-104.6 °) for 4 days longer. The plasmodia were present on Dec.
16. The temperature was normal (about 102 °) from Dec.26 to Jan. 3 but rose to104.6° on Jan. 4, 1927, and to 106° on Jan. 5. Plasmodia were found in the bloodon Jan. 3. After Jan. 5 there was no more fever. On Jan. 10, 1927, cultures of
13artonella bac~Tliformis and a saline suspension of the nodule of M. rhesus 73 wereintradermaUy inoculated. The signs of verruga were already evident on Jan.
19, and by Feb. 3 the nodules were of moderate size, and the blood had a culture
titer of 1:100. The animal had no further paroxysm of malaria and recoveredfrom both infections within a few weeks.
A n o t h e r m o n k e y was inoculated simultaneously with m a l a r i a a n dv e r r u g a material.

Protocol 3.--Macacus rhesus 3-M was inoculated on the same day as M. rhesus
2-M with 0.5 cc. of blood containing plasmodia and in addition received, bothx Mayer, M., Arch. Schiffs.- u. Tropenhyg., 1921, xxv, 150. Mayer, M.,
Borchardt, W., and Kikuth, W., Klin. Woch., 1926, v, 559; Arch. Schiffs.- u.
Tropenhyg., 1927, xxxi, 295. Noguchi, H., J. Exp. Med., 1928, xlvii, 235.
2 The parasite would appear to be Plctsmodium inui (Wenyon, C. M., Protozoology, New York, 1926, ii, 968-973).

m~xYo ~ o o v c m823

intravenously and intradermally, cultures of Bartonella baciUlformis and a suspension of verruga tissue. The animal's blood showed plasmodia on Dec. 16, 1926,
3 days before the onset of fever. The first paroxysm lasted 3 days (temperature104.6°, 106.2°, 106.2°). The second rise of temperature (to 105.4°) was first detected on Dec. 27, temperatures not being taken daily during the Christmas holidays, and fever was still present on Dec. 28 (104°). Plasmodia were present in
the blood on Jan. 3, and there was fever on Jan. 4, 5, and 6 (104°, 105.8°, 104°).
Verruga nodules arose at the sites of intravenous inoculation into the saphenousveins (where several unsuccessful punctures had been made in the course of intravenous injection), but none at the sites of intradermal injections on the abdomen.
Blood cultures made on Dec. 21, 1926, failed to yield cultures of Bartondla bacilli,formis. The temperature of 106.2°F., recorded on that day, may have been due
either to malaria or to verruga. A second injection of virulent verruga material
intradermally on Jan. 10, 1927, resulted in only slight induration at the sites ofinoculation, but the nodules on the back of the legs reached moderate size, andmarked edema of the scrotum developed. The animal recovered.
The experiments outlined gave no evidence t h a t the malarial infection in Macacus rhesus influenced unfavorably the result of infection
with Bartonella bacilliformis when the plasmodia were introduced am o n t h previous to inoculation with verruga or when b o t h parasites
were simultaneously inoculated.

Malarial Infection during Convalescence from Verruga.
T h e lesions resulting from experimental infection with BartoncUa
bacilliformis in monkeys usually heal within a few months, the microorganisms persisting longest in the spleen and l y m p h glands, wheret h e y can be demonstrated late in convalescence. I t was of interest todetermine whether, at this stage, the infection would again become
active as a result of introduction of plasmodia.
As the protocols show, the inoculation of malaria parasites duringconvalescence had no influence whatever upon the course of recovery.
In one instance the animal was reinoculated with virulent verruga
material a m o n t h after the introduction of the plasmodia, b u t reinfection was not induced. T h e malarial infection apparently had notinterfered with the development of immunity.

Protocol 4.--Macacus rhesus 4-M had had an infection with Bartondla bacilliforrais during the autumn, but the skin lesions had almost healed. It was inoculated with 0.5 cc. of the malarial blood intravenously on Dec. 10, 1926. There824

ETIOLOGY OF OROYA FEVER. XlI

was fever lasting for 6 days, Dec. 22 to 27, 1926 (temperatures varying from 104.4°
to 105°), and the blood was positive for plasmodia on Jan. 3, 1927. Blood culture
was negative for Bartonellabacilliformison Dec. 21, 1926, and the skin lesions haddisappeared.
Macacus rhesus 5-M, also recovering from verruga, was inoculated with 0.5
cc. of malarial blood at the same time as the foregoing animal. Fever began onDec. 16, 1926, and lasted 5 days (temperatures 104°-106.8°). Plasmodia werepresent on Dec. 16, 1926, and on Jan. 3, 1927. On Dec. 28, 1926, the temperaturerose to 104.4° but fell to 103.6° the next day, and there were no later paroxysms.
On Jan. 8, 1927, verruga material was inoculated intradermally. No skin lesions
appeared at the old sites, and none developed at the sites of the new inoculations,and the blood remained negative for BartanellabaciUiformisby culture. Observation ended Jan. 29, 1927.

Effect of Spleneetomy upon Verruga Peruana.
In connection with the problem of the n a t u r e of the intracorpuscular bodies known as Bartonella muris, t which appear in the red cellsof rats following splenectomy, it became of interest to determine what
effect splenectomy would have upon the course of experimental infection with Bartonella bacilliformis.
A monkey splenectomized on November 12, 1926, and inoculatedsoon afterwards with verruga material, developed the severe type ofCarrion's disease (Oroya fever), with marked anemia, extensive
edema, emaciation, and skin lesions. Even more violent reactions
than the one in this animal (Macacus rhesus 1-S) are occasionallyobserved in rhesus monkeys as a result of inoculation with Bartonella
bacilliformis,3,4 and hence the severity of the infection in this instancecannot be ascribed to the effect of splenectomy. Moreover, M.
cynomolgus 2-S, which was subjected to splenectomy and simultaneously inoculated with nodular material from M o n k e y l-S, showed amoderate reaction only. Although the blood titer was high
(1 : 100,000), there were no other evidences of systemic infection, andthe nodules were of medium size. The control m o n k e y in this instance(M. cynomolgus 5-T) reacted more severely t h a n the splenectomized
animal, although the blood titer was only 1:10, as was t h a t of arhesus control.
Noguchi, H., J. Exp. Med., 1927, xlv, 175.
4 Noguchi, H., J. Exp. Med., 1926, xliv, 697.

825

HIDEYO NOGUCH[Protocol 5.--Macacus rhesus 1-S, splenectomized Nov. 12, 1926, was inoculated
3 days later intravenously with 1 cc. of culture of Bartonella baciUiformis and 1cc. of saline suspension of nodular tissue from Macacus rhesus 2-T? The samematerial was injected intradermally. A temperature of 104° to 104.6°F. wasrecorded on Nov. 11, 24, Dec. 10, 20, and 21, and on Jan. 15, 1927. Except onthese 6 days the animal was alebrile. Blood cultures were positive on Nov. 24(1:10,000), Dec. 8 (1:100,000), and Dec. 28 (1:100). The organisms were sufficiently numerous in the blood on Dec. 3 to be detected by examination of stainedfilms. The nodules developed to moderately large size and persisted for about 2mouths. From Dec. 11, 1926, to Jan. 3, 1927, there was general edema, especiallymarked in the scrotum, and a generalized nodular eruption.
Blood Counts.
R .B.C.

Hemoglobin (Sahli)
per cent

Nov. 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nov. 24. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dec. 27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5,456,000
4,424,000
3,080,000

908050

The animal had completely recovered by Feb. 7, 1927.
M. cynomolgus 2-S, splenectomized Dec. 1, 1926, and inoculated intradermallywith a culture of Bartondla bacilliformis and a saline suspension of nodular tissue
from M. rhesus 1-S. Nodules of moderate size had developed by Jan. 14, 1927.
Blood taken on Jan. 6, 1927, yielded cultures of Bartonella bacilliformis in a dilution of 1 : 100,000. The nodules had disappeared by Feb. 9, 1927.
M. cynomolgus 5-T 5 was inoculated in the same way and with the same materialas the foregoing animal but was not splenectomized. There was no fever at anytime, but enormous cherry-red nodules developed on the eyebrows and abdominal
wall, reaching their maximum growth on Dec. 27, 1926. The blood titer was 1 : 10
on Jan. 6, 1927, but blood culture was negative on Jan. 24.

Splenectomy did not induce relapse of experimental verruga infection in two animals which had recovered (M. rhesus 40 and 4P), nor did
removal of the spleen render recovered animals susceptible to reinoculation with Bartonella bacilliformis (Iv[. rhesus 49, 50, 68). Observation continued for a period of 1 month after inoculation of Bartonella
bacilliformis into these animals failed to reveal any skin lesions, andblood cultures were uniformly negative throughout this period.

Effect of Sptenectomy and Malarial Infection Together upon Verruga
Peruana.
Two animals were subjected to splenectomy as well as to infection
with plasmodia and Bartonella bacilliformis. One succumbed to theb Noguchi, H., Y. Exp. Med., in press.

826

ETIOLOGY OF OROYA SEVIgR.

XlImalarial infection before the verruga lesions had had time to develop;
the other had a moderately severe infection with Bartonella bacilliformis and while recovering died of tuberculosis.
Protocol 6.--Macacus rhesus 6-M was splenectomized and at the same time
inoculated with 0.5 cc. of malarial blood on Dec. 10, 1926. The plasmodia were
found in the blood on Dec. 16, and 4 days later the temperature rose and remained
high for 3 successive days (104.2-106°). The second paroxysm began on Dec.
29, 1926, and lasted for 8 days (temperatures 104-106.6°). The plasmodia werevery numerous on Jan. 3. The animal was inoculated with verruga material on
Jan. 8, 1927, but succumbed to the malarial infection on Jan. 14, 1927, before theverruga lesions had had time to develop, t3artonella bacilliformis was not re-covered in blood cultures.
Macacusrhesus7-M was splenectomized on Dec. 10, 1926, and at the same time
inoculated with 0.5 cc. of malarial blood and 0.5 cc. of verruga cultures and tissue,
the latter intradermally as well as intravenously. The first paroxysm occurred on
Dec. 20 and lasted 4 days (temperatures 104.2-106°), the second, beginning J'an.
3, lasted 5 days (temperatures 104.6--106.4°). Examinations for plasmodia werepositive on Dec: 16, 1926 (+), and on Jan. 3, 1927 ( + + + ) . Blood taken on
Dec. 21, 1926, yielded cultures of Bartonellabacilliformisin a dilution of 1 : 10,000.
The animal died of tuberculosis on Jan. 10, 1927.
SUMMARY.

The experiments reported were designed to determine the influenceof malarial infection (Plasmodium inui?), splenectomy, or both combined, upon the course and character of experimental infection with
Bartonella bacilliformis in monkeys (Macacus rhesus and M.

cynomolgus).
Blood withdrawn from a monkey showing spontaneous malarial
infection was inoculated intravenously into monkeys (a) 1 monthprior to inoculation with virulent verruga material, (b) simultaneouslywith the verruga material, and (c) during convalescence from verruga
infection of moderate severity. All the monkeys contracted themalarial infection and suffered one to three paroxysms during a period
of about a month. The verruga lesions appeared in the inoculatedanimals in due course, were of average size, remained for the usual
length of time, and Bartonella badlliformis was recovered in culture
from blood which also contained the plasmodia. The lesions in theconvalescent animals continued to heal at the normal rate, and blood
cultures were negative for l~artonella bacilliformis, as is usual during

Hm~YO NOGVCm

827

convalescence. One of the recovering animals was reinoculated withvirulent verruga material a month after the injection of the malarial
blood, but neither did new lesions arise nor old ones recur. The
malarial infection, therefore, had no effect upon the course of verruga
or upon the establishment of immunity to Ba~tonella bacilliformis,
hence it would appear that malaria and verruga may coexist in thesame individual without unfavorable effect of one disease upon thecourse of the other.
Similarly, splenectomy led to no appreciable aggravation of Barlonella infection. One monkey subjected to splenectomy and inoculated with verruga material shortly afterwards had an unusually
severe reaction, but another, which was infected with material fromthe first and simultaneously splenectomized, reacted only moderately,while the non-splenectomized control showed a severer type of cutaneous infection. Even the combination of splenectomy and malarial
infection did not appreciably aggravate the experimental verruga.
Neither relapse of verruga nor reinfection with Barlonella bacilliformis
was induced in convalescent or recovered monkeys as a result ofsplenectomy.