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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
IV. T H E

E]?FECT OF INOCULATION OF /~dqTHROPOID A P E S W I T H
BARTONELLABACILLIFORM-IS.

BY H I D E Y O NOGUCHI, M.D.

(From tke Laboratories of Yke Rockefeller Institute for Medical Research.)
PLATES25 TO 28.
(Received for publication, June 18, 1926.)

It has been reported in previous papers that the intravenous inoculation of young rhesus monkeys with a strain of Barto~ella b~illiformis isolated from a case of Oroya fever gives rise to two essential
manifestations of Oroya fever, namely, protracted fever, and typical
localization of the parasite within the red blood cells (Fig. 1), while
the introduction of the organism intradermally induces skin lesions
which are indistinguishable from those of verruga peruviana. 1 It
has been shown also that after intradermal inoculation the organism
may pass into the circulation and invade the red blood cells, that it
may give rise to an anemic condition comparable with that of Oroya
fever, except for the absence of appreciable numbers of nucleated red
cells in the blood stream, and that it may induce a generalized eruption similar to that of human verruga3 In most instances, however,
the inoculated monkeys have shown little or no anemia, and comparatively few erythrocytes are invaded by the parasite, while the verruga
nodules have usually arisen only at the sites of inoculation into theskin.

Since the invasion of the red corpuscles by the bacilliform organisms
is the most characteristic sign of Oroya fever, and the formation of
nodules by the proliferation of endothelial cells is equally typical ofverruga, the findings described indicated that the organism cultivated
was probably the cause of both these conditions. Nevertheless, it*Noguchi, H., and Battistini, T. S., J. Exp. Med., 1925, xliii, 851.
~'Noguchi, H., J. Exp. Med., 1926, xliv, 697.
715

716

ETIOLOGY

O3~ O R O Y A]~EVER.

IV

was desirable to reproduce, if possible, the complete clinical pictureof Oroya fever,--the extreme anemia of pernicious type, and the invasion of large numbers ~of the erythrocytes by the microorganism,
such as takes place in h u m a n Oroya fever (Fig. 2). The opportunity
presented itself of studying the effect of the inoculation of two anthropoid apes with cultures and passage strains of Bartonella bacilIiformis,
and the experiments were undertaken in the hope t h a t these animals,
because of their close phylogenetic relationship to man, 3,4 might prove
sufficiently susceptible to the infection to manifest all of the severe
symptoms to which the parasite gives rise in man, either of the typeof Oroya fever, or t h a t of verruga. As the protocols show, however,they manifested only slight constitutional reaction to the inoculations. The erythrocytes were invaded by the organisms to a small
extent only, and there was little or no anemia. 5 The skin lesions,while of the characteristic verruga type, remained localized at ornear the sites of introduction of the microorganisms; there was no
spontaneous generalized skin eruption such as is observed in h u m a nverruga, and in rare instances in rhesus monkeys.
A young female chimpanzee (Pan leucopryrnnus), said to be about 4 years old,
was inoculated on Jan. 29, 1926, with the suspension of a nodule excised* from theeyebrow of M. rhesus 3* on the same day, and also with cultures, grown on leptospira medium, representing the second generation from the original humanblood and the first generation from the blood of M. rhesus 1.* The suspension was
inoculated intradermally into the right eyebrow and by scarification on the rightside of the abdomen, while on the left eyebrow and left side of the abdomen a
mixture of the suspension with cultures was introduced part intradermally andpart subcutaneously. The quantities injected were 0.2 to 0.3 cc. 5 cc. of the
mixture were injected subcutaneously into the right lower portion of the abdomen,
above the inguinal region, the site of inoculation being carefully massaged after
the injection.
The normal temperature of the chimpanzee, as shown by daily records coveringa period of several weeks, varied between 98.6°F. and 99.6°; occasionally theafternoon (4 p.m.) temperature was as high as 100% Blood counts made before
inoculation on Jan. 29 gave the following results: Erythrocytes 5,520,000, leuco3 Nuttall, G. H. F., Blood immunity and blood relationship, Cambridge,
1904.
* Landsteiner, K., and Miller, C. P., J. Exp. Med., 1925, xlii, 853.
5 The hematological studies were by Dr. J. H. Bauer.
* All operations were performed under ether anesthesia.

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718

ETIOLOGY O17 OROYA F E V E R .

IVcytes 7,810, hemoglobin 80 per cent. e The inoculations were made between 12noon and 1 p.m.
Jan. 30. Animal appeared as usual; appetite good. Slight irritation at thesites of scarification. Jan. 31. No change, except that the lines of scarification
were slightly raised. Feb. 3. Some swelling at the sites of intradermal injection, but no reddening. Feb. 4. Animal appeared perfectly well. Erythrocytes
5,680,000, leucocytes 8,190, hemoglobin 85 per cent.
Feb. 8. The abdominal wall showed definite edema, but the animal ate welland appeared normal. Erythrocytes 5,335,000, leucocytes 11,900, hemoglobin
85 per cent. The blood yielded cultures of Barto~Ua bacilliformis in a dilutionof 1 : 10, but routine examination of film preparations failed to demonstrate cells
containing the organism. Feb. 9. Distinct induration at the sites of intradermal inoculation on eyebrows and abdomen, but no reddening. Feb. 12.
Abdominal nodule at site of intradermal and subcutaneous injection increased
in size (3 × 5 ram.); overlying skin edematous. There was a small red spot at
the most prominent portion of the nodule. Animal still in excellent condition.
Feb. 15. Reddening of abdominal nodule had spread peripherally; a tumor of
irregular contour and uneven surface was felt under the tense, somewhat edematous skin. The tumor was firm, and the skin was partly adherent to it; there
was no softening or fluctuation on palpation. Scarified areas on abdomen showederythematous dots, but there was no definite change in the appearance of the
scarified areas on the eyebrows. The nodules induced by the intradermal injections measured 4 × 4 ram. The induration was more subcutaneous than intradermal, and at the points of needle insertion the skin was pinkish.
Feb. 19. Abdominal nodule 1.5 × 2 cm. in size, deep seated, irregular topalpation, and partly adherent to the skin, which appeared somewhat yellowish.
Abdomen less edematous. Nodules on eyebrows slightly increased in size;
overlying skin normal in color. Feb. 22. Abdominal nodule measured 5 × 6
cm. and stood out prominently above the abdominal wall. It seemed to consistof several lobulous nodules of varying size. The skin showed a definitely yellowish
tint on one side. The lymph glands in the axillary and inguinal regions and at
the flexor side of both elbow joints were markedly swollen.
Feb. 23. The abdominal nodule was harder and perhaps somewhat larger(Fig. 13). The lines of scarification on the right abdominal wall were bright
red, considerably raised, and indurated (Fig. 9). Erythrocytes 6,234,000,
leucocytes 12,000, hemoglobin 90 per cent. The blood yielded cultures ofBartonella bacilliforrais in a dilution of 1:10,000, and the organism was demonstrated in small numbers in the red cells. Feb. 25. The intradermal nodules on
the eyebrows (Fig. 8) were red at the apices. The other lesions had increased
in size and intensity.
Mar. 1. The animal had shown no constitutional reaction and appeared tobe perfectly wel. Erythrocytes 5,120,000, hemoglobin 85 per cent. Cultures of
e All the hemoglobin estimates were made with Sahli's hemoglobinometer.

HIDEYO NOGUCHI

719

undiluted blood positive. Routine examination of blood films failed to reveal
blood corpuscles containing Bartonella bacillifor~is. A portion of the abdominal
nodule (Fig. 3) was excised for examination. It showed characteristic proliferation of endothelial cells (Fig. 14), and Bartonella bacilliformis was demonstrable
in the cytoplasm of the cells (Figs. 5, 15). Cultivation of a suspension of thenodular tissue yielded growth of the organism. A portion of the skin lesion on
the scarified area of the abdomen (Fig. 9) was also excised. It was similar in
structure to the abdominal nodule (Figs. 10, 11) and yielded a culture of Barto-~lla bacilliforr~is.
For more than four weeks the blood count, temperature, appetite,and general health and disposition of the animal had remained apparently normal. There were so few microorganisms in the bloodon Mar. 1 that undiluted citrated blood in amounts less than 0.2 cc.
failed to yield growth, and demonstration of red corpuscles containing Bartonella bacilliformis was not possible b y the ordinary routine
examination of one or two film preparations. The effect of the inoculation had become mainly local, although the lymph glands were stillmuch enlarged.
At this point it seemed desirable to determine the effect of reinoculating the animal with a virulent passage strain from a rhesus monkey.
M. rhesus 14 (Text-fig. 2), which had been inoculated in the same
way as the chimpanzee and at the same time, had developed large
nodules on eyebrow and abdomen b y Feb. 23 (Figs. 16, 20), and theone at the former site had been excised on Feb. 27 for examination.
The abdominal nodule (Figs. 21, 22) was excised on Mar. 1, andsuspensions of both nodules and of a piece of skin from one of the
scarified areas on the abdomen (Figs. 18, 19) were mixed with
cultures of Bartonella bacilliformis and inoculated intravenously into
the chimpanzee. The same material was injected into M. rhesus 21
as a test of its virulence.
An accident intervened at this time.
M. rhesus 14, which had
served as the control for the first inoculation of the chimpanzee andwas showing far more active and rapidly progressing local and constitutional reactions, had been used in some experiments to determine
whether or not Bartonella bacilliformis could be transmitted by insects.
Thirty ticks (Dermacentor ar~dersoni, believed to be free from spotted
fever) were allowed to feed on the animal from Feb. 25 to Feb. 27.

720

E T I O L O G Y OF O R O Y A]FEVER.

IV

All became attached. T h e animal had been having irregular fever(104°F. or slightly above) for the past 4 weeks, and the unintentional
infection with spotted fever b y tick feeding escaped attention for atime. When the animal died on Mar. 4, however, it became evident
that d e a t h was due to spotted fever, and t h a t M . rhesus 21 (Textfig. 3) and the chimpanzee had received on Mar. 1 R o c k y Mountain spotted fever virus as well as Bartonella bacilliformis. M . rhesus
21 died of typical spotted fever within 10 days (4 days incubation,
6 days of high fever, 106°F. during the first 2 days). Notwithstanding the presence of the spotted fever infection, the blood of Monkeys
14 and 21 both yielded cultures of Bartonella bacilliformis, that ofM . rhesus 21 in a dilution of 1:100,000. Injections into guinea pigsof the blood and of suspensions of the nodular tissue from M o n k e y 14
induced typical fatal spotted fever.
Spotted Fever in the Cbimpanzee.--As the chart shows (Text-fig. 1), the spotted
fever infection (Mar. 1 to 15) gave rise in the chimpanzee to a very different
temperature curve from that of the pure infection with Bartonella bacilliformis(Jan. 29 to Mar. 1). After an incubation period of 5 days the temperature roseto 101°F., then gradually to 102.5°F., then fell to a slightly subnormal level, andremained there for several days. The fever was relatively mild, as comparedwith that usually observed in spotted fever infection in man, Macacus rhesus
monkeys, and guinea pigs; in these animals spotted fever gives rise to a temperature of 105-106° lasting 7 to 10 days. The chimpanzee had become inactiveon the 5th day, was disinclined to move about, depressed, and indifferent to food.
This condition continued for 5 days (until Mar. 9). By this time the accident
had become known through the death of M. rhesus 14, and 20 co. of pooled spotted
fever antiserum from hyperimmunized rabbits were given intravenously to thechimpanzee and also to the control rhesus (No. 21). In the rhesus the injection
had no effect; the animal died the following day. In the chimpanzee the character of the fever before and after the administration of the serum appeared tobe the same, and the recognition of curative effect is difficult unless the shortduration of the fever is assumed to have been due to the effect of the serum.
In rhesus monkeys and guinea pigs, however, the serum has not been known to
influence the course of fever unless given during the incubation period,r At all
events, the chimpanzee recovered very rapidly and probably without aid of theantiserum.
During the period of spotted fever infection, when the animal was very ill,
all the nodules, including the reddened skin lesions, became paler and smaller,
Noguchi, H., J. Exp. Med., 1923, xxxvii, 383.

mDE¥O NOGUCHI

721

and the face of the animal was cyanotic, though the color had returned withina week after the time of injection of the serum.
Mar. 1, 1 p.m. Intravenous injection of 1.5 cc. of a mixture of suspension of
nodular tissue of M. rhesus 14 (removed Feb. 27 and Mar. 1) and cultures ofBartonella bacilliformis. Mar. 5. The animal ate very little and was inactive.
The nodules on the eyebrows were still large (Fig. 12). Mar. 6. Temperature
101°F. Mar. 7. Temperature 102° a.m., 102.5 ° p.m. Mar. 8. Temperature
101 ° a.m., 102.5 ° p.m. Animal inactive, apathetic, appetite poor. The abdominal wounds, where the lesions had been removed, had partly opened, andthe surrounding tissues were edematous. Mar. 9. Temperature 102.5 ° a.m.,
102 ° p.m. The animal was very ill. The nodules were pale and cyanotic.
Erythrocytes 4,856,000, hemoglobin 80 per cent. 20 cc. spotted fever antiserum
given intravenously at 12 noon.
Mar. 10. Temperature 101° a.m., 102.5 ° p.m. The color had returned to the
nodules, and the animal was somewhat more active. Appetite improved.
Mar. 11. Temperature 102° a.m., 102.4 ° p.m. The animal was more active,
and the color of the face and nodules more nearly normal. Mar. 12. Temperature 101 ° a.m., 101.2 ° p.m. Eyelids edematous. Erythrocytes 4,288,000,
hemoglobin 65 per cent. Blood yielded growth of Bartonella bacilliformis in
dilution of 1:100. Mar. 13. Temperature 101° a.m., 101.8 ° p.m. The monkey
had removed some of the stitches from the wounds caused by excision of thelesions; the wounds were healing satisfactorily, however. Mar. 14. Temperature 100.5 ° a.m., 100.5 ° p.m. Mar. 15. Temperature 99 ° a.m., 99.5 ° p.mL
Nodules smaller and paler, not cyanotic. Erythrocytes 3,488,000, hemoglobin
60 per cent. Blood culture negative. One of the inguinal glands on the left side
was removed for examination. The suspension yielded cultures of Bartonella
bacilliformis in a dilution of 1:10. Mar. 17. Temperature 98.4° a.m., 99 ° p.m.
The animal was very active. Erythrocytes 4,344,000, hemoglobin 65 per cent.
Mar. 20. Temperature 99.5 ° a.m., 100° p.m. The remaining nodules had
diminished considerably in size and the overlying skin was of normal color.
Mar. 25 to 31. Temperature normal. The skin lesions had healed and showedslight brownish pigmentation. The site of the abdominal nodule was practically.
fiat, and the induration had disappeared. The nodules on the eyebrows werereddish but small (3 × 4 ram.).
Apr. 2. The nodules on the eyebrows were much smaller. The lymph nodes
had also decreased in size considerably. Apr. 6. Blood culture negative.
Erythrocytes 4,456,000, hemoglobin 75 per cent. May 1. The nodules hadpractically disappeared. Neither the blood nor the lymph yielded cultures ofBartonella bacilliformis. May 3. Erythrocytes 5,800,000, hemoglobin 85
per cent. The animal had completely recovered.

722

ETIOLOGY

O~'

OROYA

I~I~,V'ER.

IV

The Behavior of Bartonella bacilliformis in the Ourang-Utan.
The response of the ourang-utan to the inoculation of Bartonella
bacilliformis was similar to that of the chimpanzee, but in this instance there was a moderate febrile reaction (Text-fig. 4) which began 10 days after inoculation and lasted about a week. On the day
before the onset of fever Bartonella bacilliformis was demonstrable
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by culture in a 1:100 dilution of the blood, but a week later the titerof the blood was only 1:10. The local lesions, while characteristicof those induced by the organism, did not approach in severity thoseusually observed in rhesus monkeys. The largest nodules and theslowest to pass away were two which arose subcutaneously at thesites of attempted intravenous inoculation into the basilic vein of
the left arm (Fig. 24), and which remained prominent for 2 months.

HID~YO NOOUC~
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The ourang-utan (Pongo pygmzus), a young female (probably 2 to 3 years
old), was inoculated Apr. 17, 1926. The right eyebrow was scarified and smearedwith a piece of nodule just excised from M. rl~su~ 23 and was intradermally inoculated, on the inner aspect, with 0.2 to 0.3 cc. of a suspension of the same
tissue. 0.2 cc. of the suspension was injected intradermaUy into the shaved
skin of the right upper abdominal wall, and about 1 cc. was smeared over a scarified area on the right lower side of the abdomen. The left side of the abdomen
was inoculated similarly at two sites with a mixture of cultures derived from theblood of M. rhes~ 7 and M. rhesus 18. The mixture included cultures grown on
leptospira medium for 10 days and blood agar cultures 6 days old which conrained a large number of motile organisms.
The animal received intravenously a mixture containing 6 cc. of the cultures,
1 cc. of the suspension of nodular tissue of M. rl~sus 23, and 3 co. of citrated
blood from each of two monkeys, M. rl~sus 11 and M. rl~s~ 24. Two attempts
to introduce the syringe needle into the basilic vein of the left arm having failed,the material was inoculated into the corresponding vein of the right arm. A
small amount of material from the syringe had, however, been unintentionallyintroduced subcutaneously into the left arm, and two large subcutaneous nodules
later developed at the sites of the attempted intravenous injections.
A blood count made on Apr. 2, 1926, had shown 5,940,000 erythrocytes percomm., 12,200 leucocytes, and 85 per cent hemoglobin. The normal temperatureof the animal had been taken daily at 11 a.m. and at 4 p.m. for a period of several
weeks. I t varied from 98.6°F. to 99.6 ° and was rarely as high as 100° in theafternoon.
Blood taken on Apr. 26, 9 days after inoculation, yielded growth of Bartomlla
bacilliformis in a dilution of 1 : 100, and a few cells containing the organism were
found in film preparations. The erythrocytes at this time numbered 5,392,000,
and the hemoglobin was 70 per cent. For the 10 days following inoculation thetemperature remained within normal limits, but on the afternoon of Apr. 27 itrose to 104° , and on Apr. 28 it was 103 ° both morning and afternoon.
Apr. 29. Temperature 102.4 ° a.m., 102.2 ° p.m. Appetite poor. Lymph
glands enlarged. Slight induration at site of needle puncture on right arm.
Erythrocytes 4,880,000, hemoglobin 75 per cent. Apr. 30. Temperature 102 °a.m., 102.4 ° p.m. Animal quiet and apathetic. May I. Temperature 100.4 °a.m., 101.4 ° p.m. May 2. Temperature 101° a.m., 100° p.m. May 3. Temperature 98 ° a.m., 100 ° p.m. Diarrhea. Blood yielded growth of Bartonella
bacilliformis in dilution of 1:10. Erythrocytes 4,808,000, hemoglobin 80 per
cent. Slight induration at sites of intradermal injection on each eyebrow.
May 4. Temperature 99 ° a.m., 102.6 ° p.m. May 5. Temperature 97.4 °a.m., 99.4 ° p.m. May 6. Temperature 97 ° a.m., 97.4° p.m. Distinct nodules

724

ETIOLOGYO]? O R O Y A]?EVER.

IV

at sites of inoculation on eyebrows and areas of induration at the sites of theattempted intravenous injections on the left arm.
From this time on the temperature varied within normal limits. On May 11
the erythrocytes numbered 5,632,000, and the hemoglobin was 80 per cent.
Blood taken on May 17 yielded cultures of Bartonella bacilliforrais. The induration at the site of intradermal inoculation on the right eyebrow had disappearedby May 13, but the adjacent lines of scarification had become distinctly raised
and reddish, and there was a distinct nodule, 5 X 6 mm., at the site of intradermal inoculation on the left eyebrow. The area of scarification on the lower
right side of the abdomen also showed activity, being markedly raised and reddish. May 20. Distinct nodules on left side of abdomen at sites of inoculationof cultures. One of these was excised, and also a piece of skin from the scarified
area on the lower right abdomen. Both showed the characteristic structure oflesions induced on the skin by Bartonella bacilliformis, and the organisms were
demonstrated in sections of the tissue (Figs. 6, 25, and 26).
May 23. One of the nodules on the left arm measured 2.5 × 3 cm., the other5 X 5 ram. (Fig. 24). The nodule on the left eyebrow was about 8 X 9 mm.,and there were numerous small reddish papules on the scarified area of the righteyebrow (Fig. 23). All the lesions continued to increase in size until about
June I, when the photographs shown in Figs. 23 and 24 were taken. June 2.
Blood culture negative for Bartonella bacilliformis. Erythrocytes 5,512,000,
hemoglobin 80 per cent, leucocytes 11,600. Polymorphonuclears 33.3 per cent,small lymphocytes 45.25 per cent, large lymphocytes 3.8 per cent, large mononuclears 11.0 per cent, transitionaJs 1.0 per cent, eos~nophils 0.7 per cent, mast
cells 4.75 per cent.
The monkey scratched the nodule on the left eyebrow until on June 8 she had
succeeded in opening it and on June 10 had removed nearly half of it. The remaining portion was excised on June 10 for examination and culture (Fig. 4).
The papules on the right eyebrow had become dry and scaly at this time, andthe abdominal lesions had practically disappeared.
I n the rhesus m o n k e y , inoculated w i t h the s a m e m a t e r i a l as theo u r a n g - u t a n to serve as a control, the local reactions were less severet h a n those often observed in this species, b u t there was a tendency togeneralization of the skin lesions, and the systemic reaction was v e r ysevere. W i t h i n 7 d a y s after inoculation the sites of i n t r a d e r m a l injection on the eyebrows were definitely indurated, and there was aslight s p o n t a n e o u s eruption on the a b d o m e n . T h e t e m p e r a t u r erose on the l l t h d a y after inoculation, a n d there was continuous
high t e m p e r a t u r e (104-106°F.) after the 20th d a y (Text-fig. 5). T h ehemoglobin a t the t i m e of death, 40 d a y s after inoculation, was 15
p e r cent, a n d the e r y t h r o c y t e s n u m b e r e d 1,176,000.

mD~..Y0 N O G U C H I

725

M. rhesus 37 was injected intravenously (saphenous vein) on Apr. 17, 1926,
with the same mixture of cultures and nodular suspension which the ourangutan had received. The right eyebrow was inoculated intradermally and by
scarification with the suspension of the nodule of M. rhesus 23, the left in thesame way with the mixture used for intravenous injection. The shaved skin of
the lower right side of the abdomen was inoculated by scarification with the
nodule suspension of M. rhesus 23, the lower left side with a suspension of nodular
tissue of M. rhesus 18. The latter suspension was also inoculated b y scarification on two sites on the upper abdomen.
Apr. 23. Slight induration at the sites of intradermal inoculation on the eyebrows. Apr. 24. Indurations on the eyebrows definite; slight spontaneous
eruption on abdomen. Apr. 28. Temperature 104.2°F. Distinct nodule atsite of intravenous injection on the right leg and red papules at all four sites ofscarification on abdomen. Apr. 29. Blood yielded growth of Bartonella bacilliformis in dilution of 1:10. Erythrocytes 5,180,000, hemoglobin 70 per cent.
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May I. Temperature 104.2 °. May 3. Nodule on left eyebrow 2 X 3 ram.
May 7. Lines of scarification on right eyebrow slightly raised. Nodules onleft eyebrow and on leg increased in size. May 12. Erythrocytes 5,536,000,
hemoglobin 75 per cent. May 19. Blood culture negative.
From May 19 to M a y 24 this animal was used for the feeding of nineteen
ticks (Dermacentor variabilis). On May 20 there was a spontaneous papular
eruption on the abdomen. The nodules on the eyebrows had not increased insize, but the one on the right leg was 4 × 5 ram. On May 26, when the temperature was 105.8 ° the animal was used for the feeding of ten bedbugs, whichwere removed the following morning. May 27. Temperature 101°; animalvery ill, cyanotic. May 28. Died.
Autopsy.--Profuse hemorrhage on the abdominal wall and in the omentum
but no free blood in the peritoneal cavity. Peritoneal fluid yellowish, slightly
turbid, contained a few red cells. Intestines smooth and glistening, no evidenceof peritonitis. Lungs whitish gray, extremely anemic, but otherwise normal.

726

ETIOLOGY OF OROYA F E V E R .

IV

No fluid in the pericardium, no evidence of pericarditis. Heart muscle heavily
congested and very hemorrhagic. Liver very pale and soft but showed nogross pathological changes. Spleen normal in size but very dark in color andmuch harder than normal. Kidneys normal in appearance. There were a few
dark bluish enlarged lymph nodes in the mesenterium and one in the large intestine, but otherwise the intestinal tract appeared normal. AxiUary and inguinal lymph glands very slightly enlarged. One small nodule on the left eyebrow and another larger one on the back of the left leg. Both on section showed
the presence of BartoneUa bacillifor~nis.
Erythrocytes 1,176,000, leucocytes 3,200, hemoglobin 15 per cent. Polymorphonuclear leucocytes 50.7 per cent, small lymphocytes 31.1 per cent, large
lymphocytes 3:2 per cent, large mononuclears 11.8 per cent, transitionals 1.9
per cent, eosinophils 0.8 per cent, mast cells (basophils) 1.25 per cent, megaloblasts 1.0 per cent.
The red cells stained very irregularly, a large number of them remaining practically unstained, while about 25 per cent showed marked polychromatophilia.
There were a large number of microcytes and a few macrocytes, mostly of irregular shape. No nucleated red cells were found. A few red cells contained Bartonella bacilUformis. There were a few threads of fibrin in the smears but noplatelets.
The majority of the polymorphonuclear leucocytes had become so changed thatthe cytoplasm had practically disappeared. The nucleus was still intact. Mostof the lymphocytes were extremely small, about half the size of the red cells.
SD3~ARY.

The inoculation of a chimpanzee with cultures and a passage strainof Bartondla bacilliformis induced local reactions which, while definite
and characteristic, progressed less rapidly and were much less striking
t h a n those in the control rhesus monkey. Bartondla bacilliformis
was demonstrated in the blood corpuscles with difficulty, and thefever was slight compared with the high and persistent fever of therhesus monkey. In both the swelling of the lymph glands was an
early s y m p t o m and constantly present. Definite anemia developedin the chimpanzee only after accidental infection with Rocky Mountain spotted fever and m a y have been due to either one or both infections, though it disappeared when the blood had become negativeby culture for Bart~nella bacilliformis and the local lesions had disappeared. Incidentally, the chimpanzee was found in this one instance to be less susceptible to the spotted fever than Macacus rhesus
and guinea pigs.

HIDEYO NOGUCHI727

In the ourang-utan, also, Bartonella bacilliformis induced a mild
systemic and local infection. A rise of temperature occurred 10 days
after inoculation, and fever continued for a week, though it was
decidedly less severe than that in the control rhesus. The lesions
induced by scarification were less definite than those which arose at
the sites of intradermal inoculation. Bartonella bacilliformis wasrecovered from the blood on the 9th and on the 16th days after inoculation and from nodules excised on the 33rd and 53rd days. A few
erythrocytes containing the organism were demonstrated in stained
smears, but prolonged search was required to find them.
The symptoms and lesions observed in the chimpanzee and ourangutah as a result of infection with Bartondla bacilliformis are far milder
than those of rhesus monkeys and show less resemblance to humanOroya fever or verruga.
EXPLANATION O F PLATES.
PLATE 25.

FIG. 1. Bartanella bacilliformis in the erythrocytes of Macacus rhesus. Selected cells from film preparations of the blood of M. rhesus 25 and M. rhesus 30.
Giemsa's stain. X 1,750.
FIG. 2. Barto~ella bacilliformis in the erythrocytes of Case S. A. 15, fromwhich the organism was isolated. Giemsa's stain. × 1,750.
Fro. 3. The nodule excised from the abdomen of the chimpanzee 31 days
after inoculation. Natural size and color.
FIG. 4. The nodule excised from the eyebrow of the ourang-utan 53 days after
inoculation. Natural size and color.
FIG. 5. Bartonella bacilliforrais in a section of the chimpanzee nodule shownin Fig. 3. Giemsa's stain. × 1,750.
FIG. 6. 2~artonella baciltiforrais in a section of a nodule of the ourang-utan,
excised 33 days after inoculation. Giemsa's stain. × 1,750.
FIG. 7..Bartonella bacilliforrnis in a section of a spontaneous nodule of M.
rhesus 25 excised 23 days after inoculation. For comparison. Giemsa's stain.
× 1,750.
PLATE 26.
FIO. 8. Appearance of the nodules on the eyebrows of the chimpanzee 27 days
after inoculation. 1/2 natural size.
FIG. 9. The skin lesion on the right side of the abdomen of the chimpanzee as
it appeared 25 days after inoculation.

728

ETIOLOGY OF OROYA ~'EVER,

IV

FIO. 10. Section of the lesion shown in Fig. 9, which was excised 32 days after
inoculation. Giemsa's stain. X 182.
FIG. 11. The same section at a magnification of 1,000 times, showing thepresence of Bartonella bacilliformis.
FIG. 12. The appearance of the nodules on the eyebrow of the chimpanzee 31days after inoculation.
FIG. 13. The abdominal nodule of the chimpanzee as it appeared 25 days after
inoculation.
FIG. 14. Section of the nodule shown in Fig. 13, which was excised 32 days
after inoculation. Giemsa's stain. × 182.
FIG. 15. The same section at a magnification of 1,000 times, showing thepresence of Bartonella bacilliforn~is.
PLATE 27.
FIG. 16. Appearance of the nodules on the eyebrows of M. rhesus 14, 23 days
after inoculation. Natural size.
Fro. 17. The skin lesions on the abdomen of M. rhesus 14, 23 days after inoculation. Natural size.
FxG. 18. Section of one of the skin lesions shown in Fig. 17, excised 23 days
after inoculation. Giernsa's stain. X 182.
FIG. 19. The same section at a magnification of 1,000 times, showing thepresence of Bartonella bacilliforntis.
FIG. 20. The abdominal nodule of M. rhesus 14, 23 days after inoculation.
Natural size.
FIG. 21. Section of the nodule shown in Fig. 20, which was excised 28 days
after inoculation. Giemsa's stain. X 182.
FIG. 22. The same section at a magnification of 1,000 times, showing thepresence of Barlonella bacilHformis.
PLATE 28.
Fro. 23. Appearance of the skin lesions on the eyebrows of the ourang-utan
43 days after inoculation. 2/5 natural size.
FIG. 24. The large subcutaneous nodules which arose on the left arm of theourang-utan at the site of unsuccessful attempts to enter the basilic vein at thetime of inoculation. Photograph taken 43 days after inoculation. 2/5 naturalsize.
FIG. 25. Section of the abdominal nodule of the ourang-utan, excised 40days after inoculation. Giemsa's stain. × 182.
FIO. 26. The same section at a magnification of 1,000 times, showing thepresence of BartoneUa bacilHforrnis.

T H E JOURNAL OF E X P E R I M E N T A L M E D I C I N E

Blood of M. rhesus.

VOL. XLIV.

P L A T E 25.

Human blood.

Abdominal nodule, chimpanzee.

Eyebrow nodule, ourang-utan.

Bartonella in chimpanzee nodule.

Bartonella in ourang-utan nodule.

Bartonella in nodule of M. rhesus 25.

(Noguchi: Etiology of Oroya fever. IV.)

THE:JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 26.

(Noguchk Etiology of Oroya fever. IV.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIV.

PLATE 27.

(Noguchi: Etiology of Oroya fever, IV.)

T H E JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X L I V .

PLATE 28.

(Noguchi: Etiology of Oroya fever.

IV )