野口英世著

Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
I. CULTIVATION OF BA:RTONELLABACILLIFORM:IS.
BY HIDEYO NOGUCHI, M.D., ANDTEL]~MACOS. BATTISTINI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
P~,~s 33 xo 35.
(Received for publication, February 17, 1926.)

The microbic incitant of Peruvian Oroya fever has been recognizedby previous investigations.
Barton, t especially, detected the bacilliform "X" bodies which occur constantlyin the red corpuscles of cases of the infection and distinguished them accuratelyfrom the non-specific basophilic rods and granules present in the red corpuscles
in other diseases. Strong, Tyzzer, Sellards, Brues, and Gastiaburu, 2 who also detected the bacilliform bodies, regarded them as motile microorganisms probably of
protozoan nature which invade both red corpuscles and vascular endothelium; andthey proposed for them the name of Bartonella bacilliformis. Bartonella bacillifarmis would seem to be the specific cause of Oroya fever, as malaria plasmodia arefor malaria, despite the fact that the organism has been found in certain cases ofverruga peruviana. While this association of two clinically dissimilar diseases
has proved disturbing, Peruvian investigators had connected Oroya fever withverruga long before the bacilliform organism was discovered. According toOdriozola,a verruga may be present when Oroya suddenly supervenes, or both
maladies may arise simultaneously, or verruga may follow Oroya. These facts
have led many Peruvian investigators to regard verruga and Oroya as different
manifestations of the same disease, a conclusion supported by the heroic deed ofCarrion, who, believing in a common etiology for verruga and Oroya, submitted
himself to inoculation with verruga, the result being the development of a clinicalcase of fever, accompanied by the usual severe anemia, to which he succumbed.

We are in a relatively favorable situation today for deciding on therelationship of Oroya fever and verruga. The improvements in bac1 Barton, A. L., Cr6n. m~d., 1909, xxvi, 7.
Strong, R. P., Tyzzer, E. E., Sellards, A. W., Brues, C. T., and Gastiaburu,
J. C., Report of first expedition to South America, 1913, Harvard School of Tropical
Medicine, Cambridge, 1915.
30driozola, E., La maladie de Carrion, Paris, 1896.
851

852

ETIOLOGY

OF OROYAFEVER.

I

teriologic technique of the last few years have led to the cultivationof a number of microorganisms previously resisting culture. It was
regarded as desirable that these methods be applied to Oroya fever.
There can no longer be doubt that it and verruga can exist together,either one preceding in appearance the other. Moreover, it has beenthoroughly demonstrated4,5 that the monkey may be successfully
inoculated intradermally from cases of verruga. The possibility
exists, therefore, of testing by inoculation the power of cultures derived from cases of Oroya fever to set up the lesions of verruga inanimals.
We have applied the method originally devised for the cultivationof leptospiras e and leishmanias 7 to blood derived from a case of Oroya
fever (Santa Anna 15) in the service of Dr. Olaechea, of the Dos deMayo Hospital, Lima. Through the generous permission of Dr.
Olaechea, a blood sample was withdrawn on September 4, 1925; thepatient died 2 days later from the severe progressive anemia usual in
the disease.
Several culture tubes were inoculated with the fresh citrated l~Ioodat Lima, and these tubes, together with the remainder of the citrated
blood, were brought back to New York, the date of departure from
Lima being September 9, and of arrival in New York September 21.
The blood was kept at refrigerator temperature during the entire
period after its collection from the patient.
Bacterial contaminations occurred in the original culture tubes
during the journey, but renewed attempts at cultivation with thecitrated blood yielded a pure culture of BartoneUa bacilliformis, which
has been employed in the studies to be reported here. The media
listed in Table I were used, with the results noted.
Each tube was inoculated with 0.05 cc. of the citrated blood, placed at 37°C. for
4 days, and then removed to room temperature for 4 days or more. Only a trace
4 Jadassohn, G., and Seiffert G., Z. Hyg. u. Infectlonskrankh., 1910, lxvi, 247.
5 Mayer, M., Rocha Lima, H., and Werner, H., Miinch. reed. Woch., 1913, Ix,739.
e Noguchi, H., Y. Exp. Med., 1919, xxx, 13; Y. Trop. Med., 1925, xxviii, 185.
r Noguchi, H., Proc. Internat. Conf. Health Prob. Trop. America, Kingston,
Jamaica, British West Indies, July 22 to August 1, 1924, published in Boston,
1924, p. 455.

HIDEYONOGUCHIAND

TEL]~MACO S. BATTISTINI

853

of opalescence occurred on the surface of the tubes of leptospira medium, and they
were at first set aside as negative. On reexamination a few days later, however,numerous minute organisms were recognized. Examination by dark-field microscope, as well as staining with Giemsa's and Gram's solutions, were utilized for thedetection of the organism.
TABLE I.
1. Leptospira medium.
0.9 per cent NaC1 . . . . . . . . . . . . . . . . . .
800 parts.
Fresh rabbit serum . . . . . . . . . . . . . . . . .
100 "2 per cent nutrient agar (pH 7.4) . . . . . . 100 "Rabbit hemoglobin (made by laking
1 part of defibrinated blood with 3
parts of distilled water) . . . . . . . . . . .
10-20 "

Growth on surface layer.

2. Same, containing fresh tissue.
3. Leptospira medium containing glucose, maltose,

inulin.
4. Same, pius fresh tissue.
5. Horse blood agar slant.
Defibrinated horse blood added to melted 2 per centnutrient agar (pH 7.4) to give concentration of 20
per cent.
6. Same, containing giucose, maltose, inulin.
7. Plain agar slant.
8. Plain brofla.

No growth.
*C*¢

Bartonella bacilliformis in Culture.
Growth and Morphology.--Bartonella bacilliformis g r o w s o n solid o r
s e m i s o l i d m e d i a c o n t a i n i n g b l o o d of t h e r a b b i t , h o r s e , o r m a n ; ina fluid m e d i u m i t n e v e r m u l t i p l i e s s u f f i c i e n t l y t o b e r e a d i l y r e c o g nized. No growth can be obtained on ordinary slants or broth.
G r o w t h o n b l o o d s l a n t s is difficult t o r e c o g n i z e , since t h e colonies a r ee x t r e m e l y m i n u t e a n d t r a n s p a r e n t , h a r d l y v i s i b l e to t h e n a k e d e y e( F i g . 2). C u l t u r e s o n t h e s e m i s o l i d l e p t o s p i r a m e d i u m , w h e n t h e
n u m b e r of o r g a n i s m s i n t r o d u c e d is large, a r e m o r e r e a d i l y d e t e c t e d b y

854

ETIOLOGYO1e O R O Y A:FEVER.

I

a grayish haziness or punctiform colonies in the top layer. After 1
to 2 weeks at 28°C. the growth extends as deep as about 1 cm. below
the surface (Fig. 1, a), and resembles somewhat in appearance thegrowth of leptospiras. When, on the other hand, the number oforganisms inoculated is small, several very minute, discrete, grayish
white granules first appear, and as time goes on each colony becomes
gradually surrounded by a hazy zone (Fig. 1, b). This phenomenon
is never seen in cultures of any of the leptospiras. The organisms
grow at 37°C. as well as at 25-28°C., but at the lower temperatures
they continue to grow and survive for a much longer time. In thisrespect Bartondla bacilliformis behaves much as do various flagellates
and leptospiras, which can be most conveniently cultivated and maintained at the lower temperatures. No growth takes place underanaerobic cultural conditions.
The hydrogen ion concentration in which growth occurs lies between
pH 6.8 and 8.4, but the organisms grow best at pH 7.8. No morphological variations due to varying the hydrogen ion concentration havethus far been detected. None of the common carbohydrates (dextrose, saccharose, galactose, maltose, levulose, xylose, lactose, mannose, mannitol, dulcitol, arabinose, raftinose, rhamnose, dextrin, inulin,
salicin, and amygdalin) are fermented. Erythrocytes contained inculture medium are not hemolyzed by the growth of the organism.
When grown on blood slants at 25°C. the organisms are motile formany days and are rather uniformly rod- or spindle-shaped. Aggregates of a few to several hundred organisms are usually seen, both in
fresh preparations by dark-field illumination, and in stained ones. By
dark-field illumination, most of the young forms are seen to be actively
motile, those aggregated into masses moving along slowly, the free
individuals dashing swiftly across the field. In the leptospira medium
are found small coccobacillary forms fairly well separated from one
another when embedded in the medium. Many aggregates of organisms are also observed (Fig. 3). They are less motile in this medium
and lose their motility usually within 1 to 2 weeks at 28°C. Pleomorphism prevails, and huge abnormal spirochete-like flagella arevisible under the dark-field microscope in such cultures (Fig. 4, a).
The normal flagella of active organisms are invisible except when
stained by special methods (Fig. 4, b).

HIDEYO NOGUCHI AND TEL]~MACO S. BATTISTINI855

The organisms appear larger in fresh preparations viewed by darkfield illumination than after staining, and young forms show a definite
double contour (Figs. 3, a and b). The rods are not always straight;
sometimes they show a slight bend, and they are often massed together
in aggregates of from two or three to a hundred. In older cultures
the double contour effect is lost, the organisms appearing as decidedly
thinner rods, agglomerated into masses in innumerable numbers(Fig. 3, c). They are now no longer motile.
The microorganism is Gram-negative and stains reddish violet with
Giemsa's solution (Figs. 5 and 6). After Giemsa's staining, however,the sharp outlines are lost, and the organisms appear ill defined as if
through degeneration. Minute punctiform, spindle-shaped, or oval
elements, some in chains or in irregular entanglements, may be found.
There are also rather large and irregular forms, densely packed inmasses and taking a deeper stain. All the forms seen within the red
corpuscles of Oroya patients (Fig. 7, a, b, and c) are also to be found incultures. The stained specimens of cultures vary in length from 0.3
to 2.5u--exceptionally to 3u--and in width from less than 0.2 to 0.5~.
Chains of several individuals are also seen. Coarse oval forms measure about 0.7 by 1.2u.

Inocula2io~ Effects.
Inoculation of the cultures into various animals (ringtail, rhesus,
Java, and green monkeys, dogs, rabbits, mice, rats, and guinea pigs)
were undertaken, but the results thus far have been definite only in therhesus monkeys. The following protocols and charts represent thefindings in these animals.
Macacus rhesus 1 (Chart 1; Fig. 9). November 16, 1925, received intravenously
2 cc. of cultures from leptospira medium (9 days old) and sugar blood slant (4 daysold). At the same time the eyebrows were intradermaUy inoculated with thesame material at two sites on each side. The animal remained apparently well
until 8 days later, when it seemed less active. The temperature never rose above
104.8°F. at any time during the 55 days of observaton, the type of fever being
irregularly remittent and very mild. Areas of induration became evident at thesites of intradermal injection after 10 to 14 days, and some of these developed into
reddish nodules approximately 6 by 4 ram. in diameter.
Blood was withdrawn 13, 22, 33, and 43 days, after the inoculation. Cultures
made with the blood yielded, on each Occasion,a pure growth of Bartonella bacilli-

856

ETIOLOGY OF OROYA F E V E R .

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formis. A small number of intracorpuscular forms of the organism were demonstrated in blood smears (Fig. 8, a). One of the nodules, which was exciseds
16 days after inoculation, showed typical infiltration of mononuclear phagocytes
but was negative for Bart~ella bacilliformis. The blood withdrawn 23 days
after inoculation was transferred to three normal Macacus rhesus monkeys.
Macacus rhesus 2 (Chart 2). December 8, 1925, received intravenously 6 cc.
of a m~xture containing cultures of the 1st, 2nd, and 3rd generations derived fromthe blood of Macacus rhesus 1, as well as citrated blood withdrawn from the same
animal on two occasions (the day of inoculation and 11 days previously). At thesame time the left eyebrow was injected intradermally with the mixture and theright eyebrow with the emulsion of the nodule freshly excised from Macacus
rhesus 1. The temperature rose to 104.3°F. on the 5th day and remained at aboutthat point for 3 days. The fever in this animal fluctuated irregularly, the highest
point reached being 105°F. on the 23rd day. There were several times during the
50 days of observation when the temperature fell to subnormal. The culture madewith blood withdrawn on the 1st day of fever yielded a pure growth of Bartonella
bacillifarmis, as did also those made with blood withdrawn 9, 11, 21, 28, and 36
days after inoculation; and typical forms of the organism were found in a few of
the red corpuscles (Fig. 8, b). At the sites of inoculation on the left eyebrow
definite nodules had developed after about 28 days and these continued to increasein size during the following weeks.
Macacus rhesus 3 (Chart 3; Fig. 16). Inoculated at the same time and in thesame way as Macacus rhesus 2. The course of disease was similar to that in theforegoing animal, except that the febrile reaction was much more violent during
the first 5 weeks. I t was similarly irregular, however, and remittent. The
two sites of intradermic inoculation with culture on the left eyebrow began toshow induration within about 10 to 14 days. Only one of the two inoculations
on the right eyebrow (inoculated with the emulsion of a nodule of Macacus rhesus
1) gave rise to a nodule, and induration became evident only after 28 days, theevolution of the lesion being much slower than that of nodules produced with
culture material. All three nodules were still increasing in size when examined 45
days after inoculation. One became pedunculated and cherry-red after 2 months
and subsequently softened at the center. A yellowish fluid began to exude fromit 68 days after the inoculation.
Macacus rhesus 4 (Chart 4; Fig. 17). The date of injection and the material
injected were the same as in the foregoing two animals. The reaction was somewhat more severe than in the others, the temperature being 104°F. on the day
following the injection and reaching 105.4 ° on the 7th day. On the 16th day the
fever was interrupted, the temperature falling to slightly subnormal for 1 day.
13 days of fever followed, and the temperature was 103.4 ° on the 23rd day, when
the animal died while under anesthesia during removal of one of the nodules.
No sign of tuberculosis could be found at autopsy. The spleen was enlarged. Thea All operations were performed under ether aneshesia.

858

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ETIOLOGY OF OROYA F E V E R .

Ilymph glands were generally enlarged, but there were no lesions in the lungs.
Pure cultures of Bartonella b~cilliformis were obtained from blood withdrawn
5, 7, 11, 14, and 21 days after the inoculation, and from blood, spleen, and lymph
nodes taken at autopsy. Bartonella bacilliformis was also found in small numberin the red corpuscles (Fig. 8, c). The nodules excised 13 and 23 days after inoculation showed edema, and endothelial and capillary proliferation (Figs. 9 andD.

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CHART4. Macacus rhesus 4.
10), the microorganisms being demonstrated in cultures, smears, and sections(Figs. 13 to 15), as well as by dark-field examination of the fresh emulsion (Fig. 11).
No tubercle bacilli were found in any of the tissues examined.

Further observations on the surviving monkeys, as well as the results

HIDEYO NOGUCHI AND TEL~MACO S. BATTISTINI

861

of other animal inoculations, will be reported later. The findings here
described suffice to show that a culture of Bartonella baciliiformis
obtained from the blood of an Oroya fever patient induces in young
Thesus monkeys, when inoculated intravenously, a peculiar, irregularly
remittent, and sometimes severe type of fever, and when inoculated
intradermally into the eyebrow, gives rise to a nodule rich in cellularelements and capillary formation. Control inoculations have been
made with the culture medium employed for growing the organism,and with cultures killed by heating at 65°C. for 15 minutes. Neitherof these materials led to any of the changes induced by the livingorganisms.
Bartonella bacilliformis persists in the blood of the infected animals
for many weeks, as shown by blood cultures and smears. Intracellular forms of the organism are readily found in the proliferated
endothelial cells in the nodules, from which pure cultures are also
to be obtained.
The infection in monkeys, unlike Oroya fever in man, has not thus
far given rise to anemia, and the number of infected red corpuscles
is very small, prolonged search being required to detect one. The
lymph glands and spleen, however, become enlarged, and pure culturesof Bartonella bacilliformis have been isolated from them.
Inoculation into rhesus monkeys of the citrated human blood fromwhich our cultures were derived, after it had been kept 18 days in theice box, induced no apparent symptoms, but since the blood of theseanimals was not tested culturally the success or failure of the inoculations cannot be definitely stated. It is possible that overwhelming
numbers of young and active organisms, such as are present in cultures,are necessary to break down the normal resistance of the monkeys,
which is considerable. The relatively high resistance of such animals,
as compared with man, may explain also the fact that pronounced
anemia does not occur in the experimental infection. Further study
upon these points is necessary.
Filtration experiments with cultures have been negative.
SUMMARY.

A pure culture of a microorganism resembling in morphology andpathogenic action Bartonella bacilliformis has been obtained from blood

862

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

I

taken during life from a case of Oroya fever which ended fatally. The
blood taken at Lima into citrate solution and transported to New
York at refrigerator temperature yielded positive cultures 28 days
after its withdrawal from the patient.
The strain of Bartonella bacilliformis thus isolated grows well on
the semisolid leptospira medium, and also on slant agar containing
animal blood. The initial growth is not readily recognizable to thenaked eye, but the presence of the organisms can be determined bymeans of the dark-field microscope and by Giemsa and Gram staining
methods. No growth has been obtained on the more ordinary culture
media. The organism is an obligate aerobe, is Gram-negative, andunder certain cultural conditions motile. All the forms which havebeen described as occurring in human red corpuscles may be found in
the cultures, and in addition many granular and coarsely irregular forms
have been met with.
The inoculation of cultures of Bartonella bacilliformis into Macacus
rhesus produces infection and gives rise to effects which differ withthe mode of inoculation. The intravenous injection of the culture
into young macaques induces a prolonged irregularly remittent fever.
The organism can be cultivated from the blood over a long period,and it has been detected within the red corpuscles of the monkeys,
reproducing the precise appearances observed in human cases of Oroya
fever.
The intradermal injection of the culture into the eyebrow of young
macaques gives rise to nodular formations rich in new blood vessels
and showing the bacilliform organism within the endothelial ceils.
From the experimentally induced nodules cultures of the organism arereadily recovered.
E X P L A N A T I O N OF PLATES.

PLATE33.
FIG. 1. Appearance of growth of Bartonella bacilliforrais on leptospira medium
inoculated (a) with 0.01 co. of the citrated blood of Macacus rhesus 4 and (b) with
0.00001 cc. Fig. 1, b shows several discrete hazy colonies. Cultivation at 28°C.
for 14 days.
Fro. 2. Appearanceof surface coloniesof Barionetla bacillifoCmis on sugar blood
agar plate, cultivated at 28°C. for 4 days. The colonies were so minute and soHID]~YO N O G U C H I A N D TEL]~MACO S. BATTISTINI

863

nearly transparent that they had to be photographed by passing the light obliquelyover the plate. Hence they appear as white spots, owing to the reflection of lightby them. X 4~.
FIG. 3. Dark-field views of Bartonella badlliformis in cultures grown at 28°C.
(a) on agar blood slant for 5 days, (b) on leptospira medium for 7 days, and (c)on agar blood slant for 5 days. Organisms with definite double contour are shownin (a) and (b), while (c) illustrates the tendency of the organisms to agglomerate
into huge masses, in which it is difficult to distinguish individuals. × 1000.
FIG. 4, a. Two detached gigantic involuted flagella of Bartonella bacilliformis ina culture grown on leptospira medium for 10 days at 28°C. X 1000.
FIG. 4, b. Young organisms with flagella (6 days on blood slant at 28°C.).
Flagella stain. × 1000.
FIG. 5. Culture forms of Bartonclla bacilliformis grown on blood agar slant at
28°C. for 4 days. Giemsa's stain. X 1000.
Fro. 6. Cultures forms of Bartonvlla bacilliformis grown on leptospira medium
at 28°C. for 5 days. Giemsa's stain. × 1000.
P ~ T E 34.
FIG. 7, a to c. Bartondla bacilliformis in human red blood corpuscles. The
smear was made from the blood of the patient from whom our strain was isolated.
Several extracorpuscular forms are also shown. Giemsa's stain. × 1000.
FIG. 8. Bartonella bacilUformis in red blood corpuscles of (a) Macacus rhesus 1,(b) Macacus rhesus 2, and (c) Macacus rhesus 4, all of which were inoculated with
culture. Giemsa's stain. × 1000.
FIG. 9. Macacus rhesuz 1, showing three well developed nodules on the left
eyebrow 16 days after intradermic inoculation of culture of BartoneUa bacilU.formis.
FxG. 10. Section showing the structure of one of the nodules produced byintradermic injection of culture of Bartondla bacilliformis isolated from the bloodof Macacus rhesus 1. The nodule was removed 23 days after the inoculation.
Regaud's fixation, Giemsa's stain. × 182.
Fro. 11. Bartonella bacilliformis in an emulsion of one of the nodules of Macacus
rhesus 4. Dark-field view. × 1000.
FIG. 12. Bartonella bacilliformis in a smear of the same emulsion. Giemsa's
stain. X 1000.
FIGS. 13 to 15. Bartonella bacilliforraiS in the angioblasts and endothelial cellsof newly forming capillaries. Sections fixed in Regaud% and stained with Geimsa's
solution. × 1000.
PLATE 35.

FIG. 16. Macazus rhesus 3. The nodule on the right eyebrow developed after
the intradermic inoculation of an emulsion of a nodule excised after 16 days from
Macacus rh~us 1. The picture shows the natural size and color of the nodule 45

864

ETIOLOGY

OF OROYAFEVER.

I

days after inoculation, when it was excised for histological and cultural study andfor further passage. The two nodules on the left eyebrow resulted from the injection of cultures obtained from the blood of Macacus rhesus 1. The picture showstheir size and color 2 months after inoculation. The pedunculated nodule showsa tiny opening at the apex; it subsequently became softened at the center, and 68
days after inoculation a yellowish fluid began to exude. I t was excised the following day (February 15, 1926) for sectioning, cultural studies, and transfer.
Life size.
FIG. 17. Macacus rhesus 4. The appearance of the nodules 23 days after
intradermic inoculation of the same culture as was injected into Macacus rhesus 3.
The nodule near the median line stands out prominently and shows two roundindurations. The epidermis is expanded and shiny. There was no softening.
The nodule at the outer end of the left eyebrow was less circumscribed, but it wasmore deep seated, extending into the surrounding subcutaneous tissues. I t wasof firm consistency, like the others. The sites of inoculation, on the right eyebrow, of the emulsion of nodule tissue from Macacus rhesus 1 show scarcely a trace
of induration. Life size.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIII.

PLATE 33.

(Noguchl and Battistini: Etiology of Oroya fever. I.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIII.

PLATE 34.

(Noguchl and lqattlstlni: Etiology of Oroya fever. I.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIII.

PLATE 35.

(Noguchi and Battistini: Etiology of O r o y a fever. I.)