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ETIOLOGY

OF OROYAFEVER.

HI. Tim BEHAVIOR 017 BARTONELLA BACILLIFORMIS IN MACACUS

RHESUS.
BY HIDEYO NOGUCHI, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research.)
PraTES 22 TO24.
(Received for publication, June 12, 1926.)
As reported in a previous paper) a strain of Barto~lla badlliformis
isolated from the blood of a fatal case of Oroya fever when inoculated
into young Macacus rhesus monkeys induced a protracted course ofintermittent fever, resembling that observed in some human cases of
Oroya fever. The microorganism was demonstrated in the red cellsof these animals and recovered in culture, but the clinical manifestations in monkeys differed from those of the human disease in thatnone of the animals showed the severe anemia so characteristic of
Oroya fever. It was also shown that intradermal inoculation of thecultures into the shaved skin of the eyebrows of the monkeys gave
rise to a granulomatous nodule rich in capillaries, in which the microorganisms could be demonstrated by section and by culture. The
nodules presented a marked resemblance to those of verruga peruviana.
Further study of the behavior of Bartonella bacilliformis in rhesus
monkeys, particularly with reference to the effects of passage strains,
has brought to light a number of additional facts, which throw somelight on the variety of manifestations of t~artondla infection in man.
The protocols are presented in chronological order, and the extraordinary variability in response to inoculation of the parasite is illustrated by all of them. The severity of infection in most instances isreferable probably to enhanced virulence of the parasite throughadaptation to the animal. In Monkey 5 we have an example of severe
systemic infection induced by local inoculation. In Monkey 6 intraNoguchi, H., and Battistini, T. S., J. Exp. Med., 1925, xliii, 851.
697

698

ETIOLOGY

OF OROYAFEVER.

III

venous and intraperitoneal inoculation gave rise to a fatal systemic
infection, which was not accompanied by local lesions, and which,except for the slight degree of anemia present, resembled a case of
simple Oroya fever; the fever was severe (104-105°C.) and continuous,and the parasites were present in the blood in high titer and also in thelymph glands, spleen, and bone marrow. Monkey 7 was inoculated
intradermally and intravenously with both passage virus and cultures,and the reaction was constitutionally as well as locally severe; t~artondla bacilliformis was isolated from blood diluted 1:10,000,000, from
nodular tissue diluted 1:10,000, and also from the lymph glands,
spleen, and bone marrow. Monkey 8, on the other hand, while
receiving considerable amounts of passage virus both locally and intravenously, manifested no local and only mild systemic symptoms.
Monkey 18 is of special interest as an illustration of a severe type ofverruga such as may result from the inoculation of monkeys withhuman verruga tissues, ~. ~ while the striking fact brought out in thecase of Monkey 25 is the simultaneous occurrence of severe symptomsof both verruga and Oroya fever, typical verruga nodules appearing
spontaneously at sites remote from those of local inoculation. These
appearances had not been observed by previous investigators inmonkeys inoculated with human verruga tissues.
Quantitative estimates of the number of Bartenella bacilliformis
present at any given time in the blood are expressed in terms of thehighest dilution of the blood from which cultures of the organism
could be obtained. The culture method is the only satisfactory testof the presence of the parasite. The use of monkeys is obviously notpracticable for quantitative determination and would not, moreover,be reliable because of the variation in susceptibility to Bartonella infection. Careful microscopical examination of stained blood films wasnot, of course, neglected, but when one considers that citrated bloodof as high titer as 1 : 100,000 would show only 1 cell invaded by theparasite among 3,000 cells (1 cc. of citrated blood containing approximately 3 billion red cells), the difficulties of this method of demonstrating the organism become evident.
Jadassohn, G., and Seiffert, G., Z. ttyg. u. Infektiomkrankh., 1910, lxvi, 247.
*Mayer, M., Rocha Lima, H., and Werner, H., Mi~nch. reed. Woch., 1913,
Ix, 739.

HIDEYO NOGUCRI699

The Invasion of the Blood by B. bacilliformis after Intradermal
Inoculation.
T h e ease w i t h w h i c h Bartonella bacilliformis m a y e n t e r t h e g e n e r a l
c i r c u l a t i o n a f t e r i n t r a d e r m a l i n o c u l a t i o n is i l l u s t r a t e d in t h e c a s e ofM . rhesus 5.
M. rhesus 5 (Text-fig. 1). A young monkey, about 2,000 g m . - - a size which
has been found most satisfactory for experimental work with Bartonella bacilliformis--was inoculated, Dec. 11, 1925, intradermally on the shaved left eyebrow,with 0.2 cc. of a saline suspension of pooled cultures which on dark-field examination showed 3 to 4 motile microorganisms per field. The mixture included 1
cc. of condensation water from a 9 day old culture on a blood agar slant, derived
from the original human blood, and 0.3 cc., in 20 cc. of 0.9 per cent saline, of a
10 day old culture, derived from the blood of M. rhesus 1, and grown on leptospira medium at 25°C. Cultures made with the blood withdrawn on that date
(11 days after inoculation) revealed the presence of Bartonella bacilllformls in a
titer of 1:100,000, and in stained preparations the parasites were demonstrated
in small numbers in the erythrocytes. Cultures made on Dec. 28, 1925, andon Jan. 5, 12, and 25, 1926, also yielded positive results. The animal showed thehighest fever during this period, the temperature being continuously at 105°F.
from Dec. 29 to Jan. 1. The last two specimens of blood had a titer of only 1:10,
and the parasites could no longer be demonstrated in stained smears, hence thenumber of organisms in the blood stream had diminished rapidly within a periodof 24 days. The site of intradermal inoculation on the left eyebrow, however,began to show induration nearly 42 days after injection; on Jan. 22 it was about
2 × 3 mm. in area and was slightly raised; on Feb. 1 it measured 6 × 6 mm.,and on Feb. 4 about 9 × 9 ram. I t was at first pale and semitransparent, but
firm to the touch; then it became a deep rose-red and finally cherry-like in form andcolor. During the following 10 to 12 days it remained nearly stationary, butlater it showed slight excoriation at the apex and was removed* for transfer, culture, and histological studies. I t proved to be infective for rhesus monkeys,
yielded a pure culture of Bartonella bacilliformis, and on section showed thecharacteristic endothelial proliferation with intracellular localization of theorganism.
Blood cultures made on Feb. 9 and 17 yielded growth of Bartonella bacilliformis
in 0.1 cc. of a 1:10 dilution. At no time after subsidence of the early violent
febrile reaction had the animal's temperature been higher than 103.6°F. The
animal was killed under ether anesthesia on Feb. 17, 1926, 68 days after inoculation, in order that the distribution of the microorganisms and the character ofthe specific lesions might be ascertained.
* All operations were performed under ether anesthesia.

c

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700

HIDEYO NOGUCHI

701

Autopsy.--Spleen enlarged and granular on surface, culture +. Lymph:glands everywhere enlarged, culture +. Heart blood culture +. Bone marrow(femur) reddish, culture +. Lungs normal, culture negative. Liver normal,
culture negative. Kidney normal, culture lost because of contamination.
With the cooperation of Dr. J. H. Bauer, erythrocyte counts and hemoglobin
estimates 4 were made on several occasions. The results follow:
Jan. 12, 1926 (32 days after inoculation). Erythrocytes 6,169,000. Hemoglobin 82 per cent.
Jan. 25, 1926 (45 days after inoculation). Erythrocytes 5,901,000. Hemo.globin 75 per cent.
Feb. 15, 1926 (66 days after inoculation). Erythrocytes 5,248,000. Hemoglobin 70 per cent.
Feb. 17, 1926 (68 days after inoculation). Erythrocytes 4,856,000. Hemoglobin 70 per cent.
The chronic nature of the infection is clearly indicated by thepersistence of the parasites in the peripheral blood, bone marrow, lymph
nodes, and spleen. The number of microorganisms demonstrated bystained preparations in the blood corpuscles was never large at any
time, even during the febrile or "septicemic" period. There was little
sign of anemia until the latter part of the experiment, when the numberof red cells seemed definitely to have diminished, notwithstanding themarked fall in temperature and the scarcity of Bartonella bacilliformis
in the blood at this time.

Variability in the Effect of Inoculation of Bartonella bacilliformis into
Macacus rhesus.
The outcome of inoculation of a given pathogenic microorganism
must depend in part on the virulence of the strain and in part on the
degree of susceptibility of the host. In the case of a newly isolated
microorganism the effect of successive animal passages on virulence isproblematic; the pathogenic property may be either weakened orenhanced b y continued passage. Experiments have just been described in which a second passage of B. bacilliformis through Macacus
rhesus indicated a decided increase in virulence of the organism forthis species of animal. The present experiment has to do with theeffects of further direct passages in rhesus monkeys.
*Dr. J. H. Bauer was kind enough to make the blood counts on all the animalsof this report. The hemoglobin estimates were made by means of the Sahlihemoglobinometer.

702

ETIOLOGY O1~'OROYA FEVER.

ILl

As the protocol shows, intravenous a n d intraperitoneal inoculationof a y o u n g m o n k e y ( M . r h e s u s 6) with the citrated blood from infected
m o n k e y s of the same species led to an i m m e d i a t e and severe febrilereaction to which the animal succumbed a f t e r 3 weeks of c o n t i n u o u sfever of a b o u t 104°F.
M . rhesus 6 (Text-fig. 2) received 2 cc. of citrated blood from M . rhesus 2
intravenously on Dec. 17, 1925, and 5 cc. of a mixture of citrated blood fromMonkeys 2, 3, and 4 intraperitoneally on Dec. 18. The temperature rose to104°F. within 48 hours, dropped to 103° for I day, returned to 104°, and remained
at about that point until the 20th day. It was 103° on the 21st to 24th days
and 104° on the 25th, but on the 26th day it fell suddenly to 98° (subnormal).
The animal was cyanotic, cold, and rigid and died during ether anesthesia.
Blood cultures made on the 4th, 12th, 18th, and 26th days after inoculation
yielded the following results:
Dec. 21, 1925. Positive with blood diluted 1:100,000.
" 28, ""
(untitrated).
Jan. 3, 1926. Positive with blood diluted 1 : 100,000.
"
11, "
"
"
"
"
1:10,000.
""""" plasma "1:10.
The erythrocyte count on Jan. 3, 1926 (17th day), was 4,760,000, hemoglobin
75 per cent. Examination of organs at autopsy revealed enlargement of thespleen and of the lymph glands generally. The other organs appeared normal.
BartaneUa bacillifor~nis was obtained in pure culture from blood, spleen, andlymph nodes.

T h e striking p h e n o m e n a in the foregoing experiment were theabsence of localized lesions and the sudden collapse of the animal.
T h e latter can hardly h a v e been due to the insignificant anemia t h a t
h a d developed. T h e persistent presence of B a r t o n e l l a b a c i l l i f o r m i s inlarge n u m b e r s in the blood and organs m a y h a v e resulted in a toxemia.
T h a t different individual animals react differently to practically t h e
s a m e infective m a t e r i a l is d e m o n s t r a t e d in the following experiment.
M . rhesus 7 (Text-fig. 3) was inoculated intravenously on Dec. 21, 1925, with
5 cc. of mixed citrate blood from Monkeys 2, 3, and 4. At the same time intradermal inoculations were made on the right eyebrow with 0.2 cc. of an emulsionof the nodule from Monkey 4 and on the left with 0.2 cc. of a young culture,grown on leptospira medium, of blood from Monkey 2. Adjacent areas on each
eyebrow were scarified and smeared with the same material which had been
injected intradermally into that eyebrow.

aroEYo

Noavcm703

I n the case of this animal observation was especially directed to(1) the q u a n t i t a t i v e fluctuation of l ~ a r t o n e l l a b a c i l l i f o r m i s in the blood,(2) the course of the local nodular reactions, and (3) the effect of theinfection upon the red corpuscles and hemoglobin, this portion of thes t u d y being carried out b y Dr. Bauer.
The results of blood culture were as follows:
Dec. 24, 1925 (3 days after inoculation). Positive with blood diluted
1 : 10,000,000.

Dec. 28, 1925 (7 days after inoculation). Positive (not titrated).
Jan. 4, 1926 (14 days after inoculation). Positive with blood diluted
1 : 1,000,000.

Jan. 18, 1926 (28 days after inoculation). Positive with blood diluted 1:10.
" 29, " (39 . . . . . .
).
'. . . . . . .
1:10.
Feb. 9, " (50 """).
"(not titrated).
" 17, " (58 . . . . . .
).
"
("
" ).
T h e relatively large n u m b e r of microorganisms found in the blood
withdrawn about 72 hours after intravenous inoculation indicates a
strikingly rapid multiplication of the parasites. During the 11 days
there would appear to have been a gradual diminution in the n u m b e r oforganisms, and the findings point to an astonishing elimination in thefollowing 2 weeks, since on Jan. 18 B a r t o n e l l a b a c i l l i f o r m i s could not
be cultivated from blood in dilutions higher than 1: 10. T h e titerwas still 1: 10, 40 days after inoculation, and the organisms were present in the circulation after 58 days.
In this instance, as in m a n y others, B a r t o n e l l a b a c i l l i f o r m i s wasdemonstrable in small numbers of erythrocytes when the blood titerwas high. B u t as the fiter fell to 1:10, examination of film preparations yielded negative results.
The intradermal inoculation of the culture gave rise to a definite nodule (2× 3 ram.) within 11 days. The lesion gradually increased in size (to 4 × 6 ram.)and had assumed a rose color when it was excised for study on Jan. 8, 1925 (19thday after inoculation). No lesion developed at the site where the culture was
inoculated by scarification. The nodule at the site of intradermal inoculationof the nodule emulsion from Monkey 4 became noticeable only after 20 days, andat the same time a small area of induration appeared on the adjacent scarified
area. The nodule and indurated area became gradually redder and larger andwere removed for study 29 days after inoculation.

704

ETIOLOGY O1~ OROYA FEVER.

III

Histologically the nodules were the products of infection solely with
Bartonella bacilliformis and they showed the characteristic localizationof the organism in the endothelial cells of the lesions. An interesting
difference in the number of organisms present in the nodule and thescarified area (both induced by the emulsion of nodule from Monkey 4)
was revealed by titration. The emulsion of the skin lesion yielded
growth in a 1:10,000 dilution, while that of the nodule contained
sufficient numbers of organisms in a 1:10 dilution to yield a culture,
but not in a 1:100 dilution. It may be that the age of the lesion is afactor in this quantitative difference, since the more slowly progressing
lesion contained the larger number of organisms.
Blood counts by Dr. Bauer gave the following results:
Jan. 4, 1926 (14 days after inoculation). Erythrocytes 4,400,000.
" 12, " (22 . . . . . .
).
"4,620,000. Hemoglobin 75 per cent.
Feb. 17, 1926 (58 days after inoculation). Erythrocytes 5,472,000. Hemoglobin 80 per cent.
It is difficult to draw any conclusion from the fragmentary bloodcounts. The animal appeared, however, to be in good condition when
it was sacrificed on Feb. 17 for examination of various organs (58 days
after inoculation).
Autopsy.--The lungs showed a few scattered grayish nodules, the liver a
few small grayish spots. The spleen was enlarged and granular; the lymph
nodes moderately swollen. The kidneys were normal. Bartonella bacilliformis
was obtained in culture from the spleen, bone marrow, lymph glands, and heart
blood.

In the foregoing experiment we were apparently dealing with aprotracted and severe but non-fatal, Bartonella infection, with possible
visceral involvement. It is of special interest that the local and systemic processes proceeded simultaneously.
That an unusually resistant animal may occasionally be encounteredamong Macacus rhesus monkeys is shown by the following protocol.
M. rhesus 8 was inoculated on Dec. 30, 1925, with an emulsion of the welldeveloped nodule from M. rhesus 4.1 The material was introduced intradermally,by scarification on both eyebrows, and also intravenously (2 cc.). During 50 daysof observation there were, roughly speaking, three periods of high fever (Text-

Hm~.YO NOOUCm

705

fig. 4). During the first 12 days the temperature rose as high as 106°F., andduring the second and third periods it fluctuated about 104° for about 10 days.
Although the temperature was 104° on the day following inoculation, the febrilereaction seems to have become definite on the 6th day.
Four blood cultures were made during life and one post mortem, with thefollowing results:
Jan. 4, 1926 ( 5 days after inoculation). Positive with blood diluted 1 : 10.
....
undiluted blood.
"
8, ,, ( 9. . . . . .
).
"" blood diluted 1 : 1,000.
" 18, " (19 . . . . . .
).
"
"
"
"
1 : I0.
Feb. 3, " (35 """).
" 17, " (49 . . . . . .
).
Negative.
Blood counts showed no anemia:
Jan. 8, 1926 ( 9 days after inoculation). Erythrocytes 5,506,000. Hemoglobin 80 per cent.
Feb. 3, 1926 (35 days after inoculation). Erythrocytes 5,414,000. Hemoglobin 80 per cent.
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Feb. 17, 1926 (49 days after inoculation). Erythrocytes 5,496,000. Hemoglobin 80 per cent.
The animal was killed by etherization on Feb. 17, 1926, for examination.
Autopsy revealed marked enlargement of the lymph glands. No other changes
were noticeable. The spleen, blood, and bone marrow failed to yield cultures,but growth was obtained from the emulsion of the lymph nodes.
As a result of the inoculations on the eyebrows, four tiny indurated areas
appeared within 9 to 10 days but did not enlarge and within 18 days had practically disappeared. A red spot of pin-point size was noticed on the scarified
area after 29 days, but it also disappeared in another week.
W h i l e t h e n o d u l e e m p l o y e d for inoculation in this i n s t a n c e p r o b a b l y
c o n t a i n e d fewer o r g a n i s m s t h a n the one r e m o v e d f r o m M o n k e y 4
earlier in its evolution, y e t the suspension f r o m it yielded a p u r e
g r o w t h of B a r t o n e l l abacilliformis
in a 1 : 1 0 dilution, a n d a large n u m b e r

706

ETIOLOGY

OF

OROYA

FEVER.

IIIof organisms were found in sections. Loss of virulence may haveresulted from the final etherization of Monkey 4.
The comparatively mild infection, accompanied by severe febrile reactions, seems to have been due to a higher resistance of this particularanimal, but the chronic nature of the Bartonella infection is indicatedeven in this instance by the persistence of the microorganisms in thelymph glands.

The Reproduction of Striking Clinical Features of Oroya Fever andVerruga.
Up to this point in the work, the manifestations by monkeys ofinfection with Bartonella bacilliformis resembled human Oroya fever
or verruga in essentials, i.e., in the typical localization of the organism
and in the characteristic skin lesions, respectively, but the severe
anemia of Oroya fever and the general eruption of verruga had not as
yet been reproduced in monkeys.
In Monkey 18, however, the local lesions, while not general or
spontaneous, attained extraordinary size and resembled those described by previous investigators as resulting from the inoculationof suspensions of human verruga tissues into monkeys. Monkey 25
developed the spontaneous generalized eruption characteristic ofverruga and also severe symptoms of Oroya fever (anemia, presenceof Bartonella bacilliformis in considerable numbers in the erythrocytes). Although the period of fever was very brief in this animal,
the manifestations of infection were similar to those of certain humancases in which Oroya fever and verruga are simultaneously present3

M. rhesus 18 (Text-fig. 5) was inoculated on Feb. 15, 1926, with passage virus(suspension of nodular tissue) from Monkey 5. The shaved left eyebrow received
two intradermal injections of the emulsion, and the shaved right eyebrow wasscarified and smeared with the same material. The skin of the abdomen was
shaved, and on the left side two intradermal injections were made of the emulsion, while on the right two scarified areas (about 3 × 3 cm. each) were spreadwith the same material. Induration at the sites of inoculation became evident
in about 14 days and were definite after 26 days (Fig. 1). The lesions steadily
increased in size, and 36 days after inoculation the largest of the nodules on the
scarified areas of the right eyebrow measured 2.5 × 2 cm. and was 1 cm. high60driozola, E., La maladie de Carrion, Paris, 1896.

707

HIDEY0 NOGUCHI(Fig. 7), while the two round subcutaneous nodules on the left abdominal wallstood out about 1 cm. and showed a reddish spot at the point of insertion of thesyringe needle (Fig. 8). The lesions were of maximum size 59 days after inoculation (Fig. 2) and remained stationary for about 14 days. Retrogression was
slow, but at the time of writing (99 days after inoculation) they are pale, fibrous,and considerably decreased in size, as shown in Figs. 3 and 11. No ulceration
or softening of the lesions could be detected at any time. The examination of anodule excised from the right eyebrow 26 days after inoculation revealed thecharacteristic granulomatous structure with proliferation of endothelial cells(Fig. 9), in the cytoplasm of which were found varying numbers of B a r t o n e l l abacilliformis,
usually in clumps (Fig. 10).

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Bartonella
bacilliformis
was demonstrated in sections of the lesions and isolated
in culture from emulsions of the tissue. I t was recovered from the blood of themonkey on the following occasions:Mar. 5, 1926 (18 days after inoculation). Positive in blood diluted 1 : 100,000.
"
18,
"
(31 "
. . . .
).
""""
1 : 100,000.
" 27, " (40 . . . . . .
).
"
"
"
"
1:10.
Comparison of blood counts made on the day of inoculation and 60 days latershow that there was a definite decrease in the number of red cells during thecourse of illness:
Feb. 15, 1926. Erythrocytes 5,356,000. Hemoglobin 90 per cent.
Apr. 16, . . . .
4,120,000.
"
60 " "

708

ETIOLOGY O1~' OROYA FEVER.

III

One of the most striking features of the experimental Bar~onella
lesions is the slow continuous formation of granulomatous tissue on ascarified surface of the skin, which results in an enormous accumulation of endothelial cells and subsequent capillary formation, a process
apparently identical with that which takes place in the lesions ofverruga in man.
M. rhesus 25 (Text-fig. 6) was inoculated intravenously on Mar. 23, 1926,
with 6 cc. of a saline suspension of cultures grown on blood slants for 10 days
at 25°C. At the same time the shaved right eyebrow was inoculated intradermally as well as by scarification with a suspension of nodular tissue from M. rhesus
18. The left eyebrow was similarly inoculated with culture. This animal had~6te
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only 1 day of high temperature, Mar. 27, when 106°F. was record~l; otherwise
the course of disease was practically afebrile.
The first sign of infection in this instance was a peculiar condition of the upper
eyelids, the edges of which were irregnlarly thickened, and perhaps a little pinkish on Apr. 7, 15 days after inoculation. Within a week numerous miHaxy,cherry-red nodules appeared around the eyes on both sides, and distinct nodules
became noticeable at the site of intradermal inoculation as well as on the scarified
areas. The animal presented a striking aspect (Fig. 4). These lesions, and several miliary nodules scattered over the body, especially near the right inguinal
surface (Fig. 5) gradually became larger within the next 5 or 6 days and partly
confluent. On the posterior surface of the left leg nodules of varying size appeared within 3 weeks after inoculation (Fig. 6) and remained until death. Theeyes were virtually closed 36 days after inoculation (Apr. 28, 1926), when the
animal died (Fig. 12). One of the spontaneous nodules was removed for cultural

HIDEYO NO6UCHI

709

and histological examination on Apr. 15. It showed the usual endothelial proliferation (Fig. 13), and large numbers of Bartonella baciU@rrais were present(Fig. 14).
The number of red corpuscles infected with Bartonella baciUiformis was larger
than in any of the monkeys previously studied, cells invaded by the parasites
being readily demonstrable in film preparations. Curiously enough, however,the detection of the organism was not accomplished, even by the culture method,
during the 48 hours preceding death.
Mar. 27 ( 5 days after inoculation). Positive with blood diluted 1:1,000.
Apr. 8 (18 """).
"'. . . . .
1 : 100,000.
" 16 (26 """).
". . . . . .
1:1,000,000.
The results of blood counts by Dr. Bauer were as follows:
Mar. 27, 1926 (4 days after inoculation). Erythrocytes 5,288,000. Hemoglobin 85 per cent.
Apr. 20, 1926 (28 days after inoculation). Erythrocytes 3,240,000. Hemogiobin 45 per cent.
Apr. 22, 1926 (30 days after inoculation). Erythrocytes 2,992,000. Hemoglobin 40 per cent.
Apr. 24, 1926 (32 days after inoculation). Erythrocytes 3,368,000. Hemoglobin 40 per cent.
Apr. 26, 1926 (34 days after inoculation). Erythrocytes 2,736,000. Hemogiobin 35 per cent.
Apr. 27, 1926 (35 days after inoculation). Erythrocytes 2,120,000. Hemoglobin 30 per cent.
Apr. 28, 1926 (36 days after inoculation). Erythrocytes 1,624,000. Hemoglobin 25 per cent.
A u t o p s y . - - V e r y much emaciated, wax-yellow. The nodules on the eyebrowsremained unopened, but the lesions on the lids had partly ulcerated, and thetissues had become somewhat necrotic. The eyes were almost closed. Therewas some seropurulent discharge from the ulcerated nodules, which were nearly
confluent. Come~e not affected. Spontaneous miliary nodules were found on
the abdomen, thighs, and legs, the posterior surfaces of both legs, in particular,
being covered with cutaneous or subcutaneous nodules of varying size (Fig. 6),
some adherent to the fascia. Some nodules were hemorrhagic.
There was exudative pericarditis, due to the presence of minute Gram-negative,
motile bacilli in enormous numbers, especially within the polymorphonuclear
leucocytes. The heart muscles were pale and flabby, but the pleurae did notappear to be involved. Lungs normal. Liver perhaps enlarged, and pale.
Spleen somewhat enlarged and soft. Stomach empty, normal. Intestines andmesentery: General lymphatic system hypertrophied everywhere. Other organs
normal. Bone marrow (femur) dark red.
T h e findings described suggest a terminal secondary bacterial
pericarditis, which m a y h a v e been the result of transfer b y the animal

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ETIOLOGY OF OROYA ~FEVER.

11I

of infective material from the ulcerated eyelid to a needle woundcaused by heart puncture. Notwithstanding this late stage invasion,
the essential features of the course of illness in this animal were, I
believe, the result of local and constitutional infection with Bartonella

bacilliformis.
SUMMARY.

The experiments reported here were carried on in the main withpassage strains of Bartondla bacilliformis, and the results indicate thatthe virulence of the organism has been considerably enhanced bypassage through susceptible animals. While the animals of the earlier
experimental series showed no anemia, some of the present groupmanifested a definite reduction in the number of red ceils and in hemoglobin, and in one instance (M. rhesus 25) anemia was of the extreme
type so often associated with Oroya fever in man. The anemic condition appeared to be secondary in character, however, nucleated red
cells being few in number. In this animal also Bartonella bacilliformis
was readily demonstrated in the erythrocytes by means of stained
smears, though the number of cells invaded by the parasites was byno means so great as in the human infection.
In most instances of experimental ]~arwnella infection so far induced the demonstration of the parasites by ordinary routine examination of stained film preparations is possible only when the titer ofthe blood exceeds 1:1,000. Prolonged search of many slides has notbeen attempted, however. The number of microorganisms in the
blood, as shown by culture tests of ascending dilutions, was in most
instances highest (1:100,000 to 1:10,000,000) during the early periodof the infection coincident usually with the period of highest fever,falling to a titer of 1 : 10 during the last half of the disease. In one of
the fatally infected monkeys, however, the titer increased from 1 : 10 on
the 4th day to 1:1,000,000 on the 24th day. The titer of the blood
was equally great in Monkeys 5 and 6, although the former was inoculated locally, the other intravenously and intraperitoneally. The
largest proportion of infected red cells was found in Monkey 25, while
the blood titer, as shown by culture test, was highest in Monkey 7.
The febrile reaction varied in the animals of this series from a severe
continuous fever of 104-105°F., lasting 2 to 3 months, in one instance,

mD~YO NOGUCm

711

with a remittence during the 3rd to 5th weeks, to the acute high fever(106°F.) of 1 day's duration in the fatally infected monkey, No. 25.
The more usual reaction, however, is an irregular course of moderate
fever with one or more periods of high temperature (105°).
Bartondla badlliformis was constantly demonstrated, both microscopically and by culture tests, in the lymph glands of animals sacririced 2 to 3 months after inoculation, and in two of three instances it
was present also in the spleen, bone marrow, and heart blood. In
the case of M. rhesus 6, which died 26 days after inoculation, themicroorganism was obtained also in culture from the lymph glands,
spleen, and heart blood taken at autopsy. In the other animal which
died, a terminal bacterial infection, while not obscuring the effects ofthe t~artondla infection, made it impossible to isolate the parasitefrom either blood or tissues.
The skin lesions, whether of the nodular type, induced by introduction of the virus intradermally or by application to the scarified skin,or of the miliary character occurring spontaneously as a result ofsystemic infection, always yielded cultures of tJartondla badlliformis,and stained sections of such lesions revealed the parasites in large numbers in their characteristic situation in the endothelial cells.
A chronic, systemic infection, in which the lymph glands are enlarged and Bartonella bacilliformis is present in the blood in high titer,
may be induced by local inoculation, as shown in the case of
M. rhesus 5.
The local lesions induced in one instance by introduction of a passage
strain, both intradermally and by scarification, attained within 2months extraordinary size, the nodules arising at adjacent sites of
inoculation on the right eyebrow having coalesced into a large
pedtmculated mass which overhung the eye. This type of reaction
had not been observed hitherto in the course of the present study
but has been described by earlier investigators as a result of the
inoculation of monkeys with human verruga tissues.
The striking fact brought out in the present study is the variety of
responses to inoculation which animals of the same species may
manifest. The clinical features of the infection may be typical of
Oroya fever or may resemble those of verruga peruviana, and in
M. rhesus 25 we have an instance of a type of infection in which the

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ETIOLOGY

0~' O R O Y A F E V E R .

IIlcharacteristic p h e n o m e n a of b o t h conditions are simultaneously
present. W h e t h e r the appearance will resemble those of the one or
the other condition appears to depend on the susceptibility of theindividual as well as on the virulence of the organism. M o r e o v e r ,it seems probable t h a t different degrees of resistance to the invasionof the parasite on the p a r t of the blood cells, internal organs, or skinof a given animal m a y determine the p r e d o m i n a n t clinical m a n i festations of the infection. T h e factor of variation in susceptibilityof different individuals or different tissues of the same individual woulda c c o u n t for the v a r i e t y of types of h u m a n Bartonella infection. 6
EXPLANATION OF PLATES.
PLATE 22.
All figures natural size.
FIG. 1. M. rhesus 18, 26 days after inoculation. The nodule on the inneraspect of the left eyebrow was subsequently removed for examination.
FIG. 2. M. rhesus 18, 59 days after inoculation, when the nodules had reachedtheir maximum development. The nodules on the right side had been induced
by inoculation by scarification; there was no softening or ulceration of theselesions at any time. The nodule on the left side was induced by intradermic
inoculation.
FIG. 3. M. rhesus 18, 99 days after inoculation, when the nodules had retrogressed considerably and the animal was decidedly paler.
FIG. 4. M. rhesus 25, 23 days after inoculation. The indurations which had
developed at the sites of inoculation on the inner aspect of each eyebrow were
almost eclipsed by the numerous spontaneous deep red nodules which had appeared about the eyes.
FIG. 5. M. rhesus 25, showing the spontaneous nodules which developed in theinguinal re,on, 29 days after inoculation.
FIG. 6. M. rhesus 25, showing the spontaneous nodules which had appearedon the posterior surface of the legs 29 days after inoculation.
PLATE 23.
FIG. 7. M. rhesus 18, 36 days after inoculation, showing the appearance ofthe lesions at the sites of intradermal inoculation and of inoculation by scarification. Natural size.
FIG. 8. M. rhesus 18, 36 days after inoculation, showing the lesions on theabdomen. The verrucous lesions on the right side were induced by scarification,
6Arce, J., La enfermedad de Carrion, Lima, 1920.

Hm~:YO NOOUCHI

713

the two round subcutaneous nodules on the left side by intradermal and partly
subcutaneous inoculation. Natural size.
FIG. 9. Section of nodule removed from eyebrow of M. rhesus 18, 26 days after
inoculation. Giemsa's stain. × 182.
FIG. 10. The same section at a magnification of 1,000 times, showing thepresence of Bartonella bacilliformis.
FxG. 11. ld. rhesus 18, 99 days after inoculation, showing the appearance ofthe receding lesions on the abdomen. One of the nodules at the sites of intradermal inoculation had been removed.
PLATE 24.
FIO. 12. M. rhesus 25, at the time of death 36 days after inoculation.
Fig. 13. Section of spontaneous nodule of M. rhesus 25, removed 23 days
after inoculation. Giemsa stain. × 182.
FIo. 14. The same section at a magnification of 1,000 times, showing thepresence of masses of Bartonella bacilliformi¢.

THE

JOURNAL

OF EXPERIMENTAL

FIG. l. M. rhesus 18,
26 days after inoculation.

MEDICINE

VOL. XLIV.

PLATE 22,

FIG. 4. M. rhesus 25,
23 days after inoculation.

FIG. 5. M. rhesus 25(groin), 29 days after inoculation.
FIc,. 2. M. rhesus 18,
59 days after inoculation.

FiG. 3. M. rhesus 18,
99 days after inoculation.
FIG. 6. M . rhesus 25(leg), 29 days after inoculation.
(Noguehi : Etiology of Oroya fever. III.)THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X L I V .

PLATE 2,:I.

(Noguchi: Etiology of Oroya fever. III.)THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 24.

(Noguchi: Etiology of Oroya fever. III.