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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
X I H . CttEMOTIIERA.PYIN EXPE]~ rm~.NTALBARTONELLA BACILLIFORMIS

I.I~ECTIOI~.
BY HIDEYO NOGUCIII, M.D.
(From tM Laborahr~s of The Roakefdler I~titut¢ for Medical Rt3earch.)
PLATES15 AXD 16.
(Receivedfor publication, January 13, 1928.)
In the course of our studies on Bartonella bacilliformis infection inmonkeys, we submitted several Macacus rhesus, in which experimental
verrucous lesions had been induced by means of cultures, to treatment
with chemicals which had proved therapeutically useful in spirochetal
and leishmania infections. Salvarsan had already been recommended
by Arcet in the treatment of malignant verruga in man. In ourexperiments salvarsan, neosalvarsan, bismuth lactate, esters of chaulmoogra oil, sodium gynocardate, neutroflavine, and urotropin weretested. It was not deemed suitable to test tartar emetic, since it isa drug of slow therapeutic action, and the experimental verrucous
lesion~ in the monkey lead in any case to spontaneous retrogression.

Chemical Action in Vitro.
Although it was not expected that a relationship would be shown toexist between the action of the chemical when tested on ]Bartondla invitro and the verrucous lesions in vivo, it was considered of interest to
determine the direct effects of the chemicals on the bacilli. The
substances to be tested were added directly to the culture media, andthe cultures were incubated at 25°C. for a period of 13 days. The
results are shown in Table I. Neutroflavine inhibited growth in
1:10,000,000 dilution. Formalin was almost as effective, and neosalvarsan, novasurol, and mercuric chloride were effective up tot Arce, J'., An. Favultad Med. Lima, 1918,i, No. 3, 21-53, 130-161; No. 4, 24-52.
619

620

E T I O L O G Y OF OI~OYA ~FEVER.

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1:100,000. Mercurochrome and tartar emetic required at least
1:10,000 concentration to prevent growth.
The first experiments were made on monkeys in which the cherry-red
verrucous lesions on the abdominal skin and eyebrows had reachedmaximal size and had persisted in this state for several days. Bloodcultures taken shortly before or at the time of first injection of thechemical into the circulation proved subsequently to be negative,
and bits of excised nodules taken at the same time 2 showed few bacilli
or even none at all by culture or in section. These last findings could
not be known at the time of treatment, since the bacilli require 10 to
14 days to become evident in culture.
However, distinction between the ordinary or spontaneous regressionof the nodules and the regression taking place after the use of chemicals, is entirely possible. The mature nodules undergo spontaneous
regression slowly,3 while in the animals given chemicals there occurred
rapid loss of cherry-red color, usually in 24 to 48 hours after the first
injection, followed by a still more rapid reduction in size most pronounced in the nodules located in the abdominal skin. At the expiration of 5 to 6 days small pale fibrous areas alone remained to indicate
the site of the nodules, and in 10 to 14 days all vestiges had disappeared,
the lesions of the eyebrows persisting somewhat longer than those of
the abdomen. The protocols of these experiments follow.
Protocols.
Macacus rhesus l-T, injected intravenously on Oct. 14, 1926, with 0.5 cc. of a
mixture of:
4 cc. defibrillated blood (culture + + +) from M. rhesus 54 (P. 5 strain4),
5 cc. culture of Bartondla bacilliformis (P. 5 strain) grown on leptospiramedium for 72 hours,
5 cc. culture of Bartonella bacilliforrai's (P. 5 strain) grown for 6 days on blood
agar slants.
In addition, 4 intradermal injections of the mixture were made on the left
abdominal wall and 2 on the left eyebrow. Also nodular tissue freshly excisedfrom M. rhesus 54 was applied to scarified areas on the right abdominal wall andright eyebrow.
Ether anesthesia was used in all the operations.
3 Noguchi, H., J. Exp. Med., 1927, xlv, 455.
4 Noguchi, H., Y. Exp. Med., 1927, xlv, 175.

622

ETIOLOGY OF OROYA :FEVER.

XlII

Experimental nodules appeared on the left eyebrow in 14 days. Within amonth large nodules were present on the abdomen at the sites of intradermalinoculation, aud the scarified areas showed the characteristic millary lesions. OnNov. 23 (40 days after inoculation) the hemoglobin was 35 per cent (Sahli), and thered cells 2,502,000, and there had been no fever. 0.05 gm. of salvarsan was given
intravenously. Within the following week the nodules grew small and became
paler. On Dec. 3 a second injection of 0.05 cc. salvarsan was administered.
Within the week the nodules had become very small and pale, the erythrocytesrose to 5,600,000 and the hemoglobin to 80 per cent. When the animal was
sacrificed on Dec. 13, Bartonella bacilliforrMs could not be detected in the nodular
tissue either microscopically or by culture, and Mood, lymph nodes, and spleenalso failed to yield cultures. Sections of the nodules showed fibrous tissue.
Macacus rhesus 2-T. Inoculated at the same time as No. l-T, and in the samemanner, except that the eyebrows were spared. On Oct. 19 the culture titer ofthe blood was 1 : 100,000. On Nov. 15, or 32 days after the inoculation, the animal
showed large mature subcutaneous nodules on the left abdominal wall and numerous red miliary lesions on the scarified areas on the right side (Fig. 1). Bloodcultures made on this day later proved to be negative. A mixture of 1 cc. of 1
per cent bismuth lactate, 1 cc. of i per cent neutroflavine, and 1 cc. of 1 per centurotropin was injected intravenously. The temperature rose to 105.2°F. on thefollowing day (Nov. 16), but the animal appeared well. On Nov. 23, a second
injection of the same mixture was given. The nodules soon became bluish,
smaller in size, and continued to diminish in volume after the second close of thedrugs (Fig. 2). On Dec. 3 a third injection was given. During the following 9days the nodules became very small and pale (Fig. 3). Nodules and spleen
removed on Dec. 16 did not yield Bartonella bac,'lli]ormis in culture.
Macacus rhesus 3-T, inoculated in same manner as Macacus 2-T. By Nov. 24
the abdomen showed large, mature subcutaneous nodules (Fig. 4), and large
bluish red nodules on each leg where the injections had been made into the saphenous veins. A general miliary eruption was also present on the abdomen (Fig. 5).
The hemoglobin was 45 per cent (Sahli), and the red blood cells 3,960,000. No
fever. A suspension of one of the nodules yielded cultures of Bartonella bacillifermis in a dilution of 1:1,000, but the blood proved negative, although the titer
had been 1 : 100,000 on Oct. 19 mad Nov. 3. At this time (i.e., on Nov. 24, 4i days
after inoculation), 1 cc. of moogrol5 was given intravenously. Within 48 hours thenodules had become bluish in color and somewhat smaller. Fig. 6 shows theappearance of the nodules 9 days after the first treatment. On Dec. 3 anotherinjection of 1 cc. of moogrol was given. The temperature rose on the 2 days
following to 104.4--104.6°F. By Dec. 13 the nodules had contracted considerably(Fig. 7). The spleen was negative for culture on Dec. 16, and all lesions had
disappeared by Dec. 30. The blood culture was negative a week later.
s Burroughs Wellcome and Company's preparation of the esters of chaulmoogra oil.

mI)~.YO NOGVCHI623

Macacus rhesus 4-T was inoculated intravenously on Nov. 24, 1926, with 1
cc. of a mixture of cultures of Bartonella bacilliformis, besides which a suspension ofan abdominal nodule of M. rhesus 3-T was given intravenously and introduced
intradermally into both eyebrows and the abdominal skin. On Dec. 21 the blood
cultures were positive for Bartonella bacilliformis in a 1:100,000 dilution. The
nodules were fully developed by Dec. 28, 34 days after inoculation (Figs. 8 and11), when the first intravenous injection of bismuth lactate, proflavine, and urotropin was given (a mixture of 1 cc. of a 1 per cent solution of each). Bloodcultures were negative at this time. The temperature rose to 105°F. on the day
following the treatment, and the nodules had already shrunk and become paler.
A double dose was given on Dec. 30, and again on Jan. 3, 1927. No febrile reactions. Blood culture was negative on ]'an. 5, 1927. The hemoglobin was 78 percent (Sahli), and the erythrocytes 4,400,000. Cultures made from nodules andlymph nodes on ]'an. 10, 1927, were negative. The lesions regressed rapidly(Figs. 9 and 12), and only fibrous traces remained on ]'an. 28, 1927 (Figs. 10and 13).
Macacus cynar,~olgusS-T, inoculated intradermally with a suspension of nodular
tissue from M. rkesus I-S° into the right eyebrow and the abdominal skin on
Dec. 7, 1926. This animal remained afebrile but developed large cherry-red
nodules on eyebrows and abdomen by Dec. 27. The blood culture titer was 1:10on ]'an. 6, 1927. On Jan. 8 and 14, 1927, or 32 and 38 days after inoculation, an
intravenous injection was made of 1 cc. of 5 per cent bismuth lactate, 1 cc. of 5
per cent urotropin, and 1 cc. of 1 per cent neutroflavine. Figs. 14 and 15 show
the appearance of the lesions on Jan. 7. The nodules began to shrink in thefollowing week and within 2 weeks had become small and fibrous. The appearance on Feb. 8 is shown in Figs. 16 and 17.
T h e n e x t step was to test the action of the chemicals on the a p p e a r ance a n d d e v e l o p m e n t of the nodules in instances in which the chemicals were a d m i n i s t e r e d before the lesions reached full d e v e l o p m e n t ,i.e., 2 to 3 weeks a f t e r the inoculation of the infective m a t e r i a l into
the skin, while the lesions were growing in size daily. U n d e r thesecircumstances the drugs failed to influence the progress of the lesions,which in one instance a t t a i n e d unusually large p r o p o r t i o n s (Figs.
19, 20). T h e usual cherry-red color developed w i t h o u t hindrance, a n d
the scarified areas became covered with the characteristic deep red
miliary nodules. As in u n t r e a t e d animals, the skin of the lower halfof the a b d o m e n became edematous.
e Noguchi, H., J. Exp. Med., 1928, xlvii, 821.

624

ETIOLOGY OY OROYA FEVER.

XlII

Protocols.

Macacus r~sus 6-T, inoculated intravenously (2 cc.) and intradermally on
Dec. 28, 1926, with a mixture of cultures of Bartondla bacilliforr,,is and a suspension of nodular tissue of M. rhesus 4-T. The nodules were well advanced on
Jan. 24, 192;', 27 days after the inoculation, when intravenous injection of a
mixture of bismuth, urotropin, and proflavine (1 cc. of 1 per cent proflavine, 1 cc.
of 5 per cent urotropin, and t cc. of S per cent bismuth lactate) wasbegun. Bloodtaken just before the treatment yielded cultures in a dilution of 1 : 10. No change
was observed in the nodules after the first injection. The second injection, givenon ~'an. 26, 1927, was fonowed by slight diminution in the size of the nodules
on the eyebrows while the abdominal lesions continued to enlarge.
Macacus rhesus 7-T, inoculated by scarification and intradermal injection onMar. 8, 1927, with a suspension of the nodule from M. rhesus 3-A, which had been
infected by means of the L5 strain of Bartonella bacilliformlsY Small nodules werepresent 20 days after inoculation (Fig. 18), when the animal was given the first
intravenous injection of 0.1 gin. of neosalvarsan. The injection was repeated 2
days later. The nodules continued to grow gradually and within 2 weeks theyattained unusually large dimensions (Figs. 19 and 20). Certainly no inhibitory
action was apparent.
Macacus rhesus 8-T. This animal was inoculated in the same manner and on
the same date as the foregoing monkey. Nodules had appeared by Mar. 28(Fig. 21), when an intravenous injection of 2 cc. of 3 per cent sodium gynocardate
and an intramuscular injection of 0.15 cc. of chaulmestrol s were given. This wasfollowed 2 days later by an injection of 3 cc. of the sodium gynocardate and aninjection of 0.2 cc. of chaulmestrol. No inhibitory effect upon the developmentof the cutaneous lesions was apparent and the large nodules are shown in Figs.
22 and 23.
SUMMARY.
T h e t h e r a p e u t i c effect of several a n t i p a r a s i t i c chemicals on experim e n t a l v e r r u g a p e r u a n a is described. T h e drugs were a d m i n i s t e r e d b yi n t r a v e n o u s injection a c c o r d i n g as t h e n o d u l e s (1) were a l r e a d y dev e l o p e d to an a p p r o x i m a t e m a x i m u m , or (2) were still in the a c t i v eperiod of g r o w t h .
T h e effect of the drugs w a s different u n d e r t h e two c i r c u m s t a n c e s o f
Noguchi, H., Y. Exp. Med., 1928, xlvii, 219.
s The name given by the Winthrop Chemical Company to their preparation of
the esters of chaulmoogra oil, of which the Company kindly furnished a sample.

mOEYO NOOVCm

625

their administration. When they were given after the maturity of thenodules they hastened the regressive process, but when given during
active growth of the lesions no action whatever was detected.
t~artonella bacilliformis in culture is acted upon injuriously by anumber of the chemicals employed in the therapeutic tests, the mostactive being formalin and neutroflavine.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X L V I I I .

PLATE 15.

t"qBefore treatment (32 daysafter inoculation).

17 days after treatment. 25 days after treatment.
(One nodule removed for study 32 days after inoculation.)t~

Before treatment. Nodular and miliaryeruptions 41 days after inoculation.

Beforetreatment.
Nodules on eyebrows andabdomen 34 days afterinoculation.

9 days after treat- 19 days after treatment.
ment.
(On~ nodule removed for study 41 days
after inoculation.)

13 days after treatment. 38 days after treatment.
(One nodule removed for study 34 days after inoculation.)(Noguchi: Etiology of Oroya fever. XIII.)THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E V O L X L V I I I .

Before treatment (32 days after inoculation).

PLATE 16.

31 days after treatment was begun.

Before t r e a t m e n t (20days after inoculation).

15 days after treatment was begun.

Before treatment (20 daysafter inoculation).

8 days after first treatment.

15 days after treatment
was begun.

(Noguchi: Etiology of Oroya fever. XIII.)