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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
]~. COMI~ARATIVE STUDIES OF DIFFERENT STRAINS OF BARTONELLABACILLIFORMIS, WITH SPECIAL R~EFEP~ENCE TO THE RELATIONSHIP BETWEEN THE CLINICAL TYPES OF CARRION 'S
DISEASE AND THE VIRULENCE OF THE IN~ECrI~G

ORGANISM'.
BY H I D E ¥ O NOGUCHI, M.D.

(From tke Laboratories of The Rockefeller Institute for Medical Researck.)
PLATES 7 TO I0.
(Received for publication,November 5, 1927.)

Dr. Sebastian Lorcnte, Director of the National Department of
Public Health of Peru, during a visitto N e w York in 1926, expressed
the wish that experimental studies of Carrion's disease might befurther extended and agreed to forward specimens of patients' bloodto The Rockefeller Institute for the purpose. Six samples of blood
were received from Dr. Lorente on December 29, 1926, and six moreon April I, 1927. Comparative studies of the strains of Bartonella
bacilliformisisolatedfrom these spocimens form the subject matter ofthe present report. I wish at the outset to express to Dr. Lorentem y appreciation of the coopcration which made the work possible.

Isolation o/Bartonella bacilliformis.from Specimens of Patients' Blood.
The blood was collected in sterile vacuum bulbs containing dry
sodium citrate sufficient to prevent coagulation (0.1 gin. per 10 cc. ofblood). The containers were hermetically sealed and shipped in thesteamer's refrigerator from Lima to New York, the time required fortransportation being about 14 days. Three of the first six samples
were unsuitable for cultural study, having become contaminated withextraneous bacteria by leakage of the container during transportation.
The second shipment (Cases 7 to 12)arrived in perfect condition.
BartoneUa baciUiformis was obtained in pure culture from all six
219

220

ETIOLOGY

OF

OROYA

FEVER.

X

specimens of the second lot a n d f r o m the three u n c o n t a m i n a t e dsamples of the first lot, nine strains in all being isolated.
T h e blood was inoculated into leptospira m e d i u m b y the t i t r a t i o n
m e t h o d , t h a t is, several dilutions were m a d e of each specimen (1: 10,
1: 100, 1: 1,000, 1: 10,000, 1 : 100,000), a n d a t u b e of culture m e d i u m
was inoculated for each dilution a n d one for the undiluted citrated
blood, a t o t a l of six t u b e s of m e d i u m for each sample of blood. T h e
a m o u n t of inoculum was 0.2 cc.
Dark-field examination revealed no motile organisms. I n stained
smears f o r m s resembling t~artonella were found, b u t t h e y were n o t
sufficiently n u m e r o u s or characteristic to be identified with certainty.
Case 1, F. A., 18 years old, resident of Canta, Department of Lima. Had
been ill 3 months. Verruga suspected because of patient's residence and his
symptoms. Erythrocytes 2,900,000. Leucocytes 7,000; neutrophils 72 per cent,eosinophils 1 per cent, monocytes 7 per cent, lymphocytes 20 per cent; no basophils. Ptas~nodiurnfalciparur~ was present in the blood. Temperature 37.8°C.
at time blood was taken.
Case 2, J. M., 45 years old, resident of Oroya, Department of J'unin. Had
been ill 2 months. Generalized miliary verrucous eruption as well as subcutaneous nodules. There had been fever of remittent type, but patient wasafebrile at the time of taking blood. Erythrocytes 2,500,000. Leucocytes,
13,600; neutrophils 65 per cent, eosinophils 2 per cent, basophils 1 per cent,monoCytes 10 per cent, lymphocytes 22 per cent.
Case 3, G. H., 20 years old, resident of Matucana, Department of Lima. Had
been ill 15 days. Verrucous eruption of miliary type, scattered. Fever ofremittent type still present,, highest temperature 40°C. Erythrocytes 3,400,000.
Leucocytes, 9,000; neutrophils 69 per cent, eosinophils 1 per cent, monocytes
8 per cent, lymphocytes 22 per cent; no basophils. Barton's bodies present,
bacilliform type. Wassermann reaction positive.
Case 4, L. W., 37 years old, resident of Oroya, Department of Lima. Had
been ill 1 month. Verrucous eruption of subcutaneous miliary form, scattered,localized chiefly in lower limbs. Patient had no fever at time blood was taken.
Case 5, A. F., 31 years old, resident of Chosica, Department of Lima. Had
been ill 4 months. Miliary eruption and subcutaneous nodules were present on
both arms and on thorax. Fever of remittent type still present; highest temperature 39.5°C., temperature at time blood was taken 37.8°C. Erythrocytes
1,920,000. Leucocytes 4,600; neutrophils 40 per cent, eosinophils 3 per cent,basophils 1 per cent, monocytes 9 per cent, lymphocytes 47 per cent. Wassermann reaction positive.
Case 6, F. T., 28 years old, resident of Matucana, Department of Lima. Had
been ill 2 months. No eruption at time blood was taken, but for 1 month patient

HIDEYO NOCUCHI

221

had subcutaneous miliary lesions. Erythrocytes 2,460,000. Leucocytes, 6,400;
neutrophils 73 per cent, eosinophils 2 per cent, monocytes 2 per cent, lymphocytes 23 per cent; no basophils.
Case 7, S. N., 22 years old, resident of Puruhuay (Chosica), Department of
Lima. No fever. Generalized verrucuous eruption of miliary and nodular
types. Erythrocytes 3,480,000. Leucocytes 4,400; neutrophils 72 per cent,eosinophils 1 per cent, basophils 1 per cent, monocytes 4 per cent, lymphocytes
22 per cent.
Case 8, J'. A., 33 years old, resident of Canta, Department of Lima. Had beenill 6 months. No fever. Generalized miliary eruption. Erythrocytes 3,100,000.
Leucocytes, 4,000; neutrophils 65 per cent, eosinophils 1 per cent, monocytes 8
per cent, lymphocytes 26 per cent; no basophils.
Case 9, J'. M., 19 years old, resident of Chosica, Department of Lima. Had
been ill 15 days. Continuous fever, 39°C. No eruption. (Malignant Verruga?)
Erythrocytes 1,250,000; normoblasts 4 per cent, erythroblasts 2 per cent.
Anisocytosis, anisochromia, polychromatophilia. Leucocytes 8,400; neutrophils
73 per cent, eosinophils 2 per cent, monocytes 3 per cent, lymphocytes 22 per
cent; no basophils. Barton's bodies present, bacilliform type predominating.
Jolly bodies also present.
Case 10, E. C., 39 years old, resident of Chosica, Department of Lima. Had
been ill 1 month. No fever. Verrucous eruption, miliary and nodular, scattered.
Erythrocytes, 5,080,000. Leucocytes 5,200; neutrophils 44 per cent, eosinophils
7 per cent, basophils 1 per cent, monocytes 7 per cent, lymphocytes 41 per cent.
Barton's bodies of coccoid type present.
Case 11, M. C., 27 years old, resident of Chosica, Department of Lima. Had
been ill 12 days. No fever, and no eruption at time blood was taken. Erythrocytes 1,080,000; normoblasts 9 per cent, erythroblasts 2 per cent, neutrophilic
myelocytes 3 per cent. Megalocytes. Leucocytes 22,200; neutrophils 88 per
cent, monocytes 3 per cent, lymphocytes 9 per cent. No eosinophils or basophils.
Barton's bodies, coccoid form, present.
Case 12, M. L., 28 years old, resident of Matucana, Department of Lima.
Had been ill 1 month. No fever. Eruption of miliary type, discrete. Erythrocytes 4,800,000. Leucocytes 4,900; neutrophUs 54 per cent, eosinophils 5 per
cent, monocytes 10 per cent, lymphocytes 31 per cent; no basophils. Barton'sbodies present, coccoid form predominating.

Comparative Studies of the Strains Isolated.
A ppearat~ce of Growth (30°C.).
On leptospira medium there is a light grayish, usually homogeneous, butsometimes finely granular, translucent layer of growth at the top of the columnof medium. Single isolated colonies form denser central masses and are surrounded by a hazy zone of outgrowth from the periphery. All the strains pre-222

ETIOLOGY OF OROYA FEVER.

X

seated a similar appearance except Nos. 9 and 12, which grew faster and more
densely than the others. Horse blood agar plates or slants yield minute grayish,raised, shiny, firm miliary colonies which appear transparent but are grayish
when covered with a layer of saline solution. They adhere to the medium andare difficult to scrape from the surface. Strains 9 and 12 form somewhat larger
colonies, which reach a diameter of 2 nun. within 4 or 5 days.

Motility (Dark-Field Observations).
Cultures on leptospira medium or horse blood agar slants or plates containvery actively motile forms for the first 4 to 6 days, especially in the condensationwater of the blood slants, but the organisms invariably become motionless sooner
or later. The flagella apparently become abnormally large under the unfavorable
conditions existing in a semisolid medium and are thrown off; for detachedflagella, so large as to be visible by dark-field examination, appear in cultures
about a week old (Figs. 32 and 33).
I have been unable to detect any differences in movement among the ~trainsstudied; they all exhibit rotation and progression, singly or in groups of two or
three, or in masses. Occasionally there is no motility at all even in young cultures, owing undoubtedly to unfavorable conditions of cultivation.

Staining Properties and Dt,imensions.
All the strains are frankly Gram-negative and take basic fuchsia rather poorly.
Giemsa's solution gives satisfactory results and brings out the fairly sharp contours of the organisms in cultures which have been grown 4 to 6 days on thesurface of blood agar slants or plates. This definition of the form is quickly
lost as the cultures grow older. Organisms grown on leptospira medium or in
the condensation water of a blood agar slant stain only poorly, and it is often
impossible to recognize individuals; nevertheless they thrive well and remain
viable under these conditions much longer than on the surface of slants or plates.
There is a tendency for most of the strains to take up the stain more intensely in
the interior portion of the body than on the outer layer. Often the chromatophilic material is seen to be heaped up toward either end, giving the appearanceof a diploid coccobacillus. Sometimes the accumulation is in nodular masses
at irregular intervals throughout the entire length, the effect simulating a chainof ill stained cocci, while in shorter forms there may be only a single deeply stained
dot. The organisms often assume a kind of triangular or elongated wedge shape.
I n fresh preparations of young, actively motile cultures, the organisms all
appear to be of the same size and shape, but certain variations in size and form
are recognizable in stained smears (Plates 7 and 8). Special care has beentaken to exclude various external factors which might influence the comparison.
For example, cultures have been grown on the same medium (horse blood agar
plates prepared on the same day with the same material) for 6 days at 30°C.

HIDF.YO NOGUCHI

223

under identical conditions, and film preparations have been made in the sameway (the culture put into a drop of distilled water, the mixture spread on theslide, and dried in the air for a given period), fixed in methyl alcohol for 5 minutes,
and then stained with Giemsa's solution in the same jar for 30 minutes. Anotherset of slides has been treated by Gram's method and counterstained with dilutecarboI fuchsin, washed, blotted, and dried in the air. The measur~acnts given.
in Table VI, therefore, should be fairly reliable. Strain 7 (Figs. 5 and 6) isdistinctlycoarser and Strain II (Figs. 13 and 14) decidedly finer than the rest.
There is, however, a gradual transition, some approaching the coarser typeand others the finer, hence striking differencesare brought out only by comparing the two cxtremes. Some strains are short, others more rod-shaped.

Flagella (Zettnow-Fontana Combination Stain).
Determination of the type of flagella in newly isolated pathogenic microo~gauisms is not of morphological interest merely; it may prove to be of
immunological importance. Orcutt 1has demonstrated that the serological specificity of certain organisms resides in the flagella, and Bauer~ recently foundin this laboratory that serological types of tetanus bacilli possess dissimilar flagella; for example, Type III, having a single unipolar heavy spiral flagellum, andType IV, having peritrichal, rather coarse regularly wavy flagella, of moderate
length, are quite distinct from the other types. All strains of Bartonella bacilliformis were therefore stained for flagdla by a modified procedure, in which
Zettnow's mordant is followed by reduction with Fontana's ammoniac sUver
nitrate. The cultures used were those utilized for the measurements of size, thatis, the surface growth on horse blood agar plates after 6 days at 30°C.
Certain strains proved to have comparaSRvdy shorter, smoother, and moredelicate flagella than others (Plate 9). Strains 3, 9, 10, 11, and 12 (Figs. 22,
26 to 29) are of this type, whiIe StrUms 2, 7, 8 (Figs. 21, 24, and 25), and the two
old strains, S.A.8 and P.5 * (Fig. 30) have distinctly more wavy or spiral andcoarse appendages. The number of flagella seems to vary among different
strains, some showing a single flagellum, others as many as four. In all instances,, however, the flagella are unipolar. Short tlageUa scarcely exceed 3~,
long ones may measure 10n or more.

P atkogen,ici~y.
Macacus rhesus monkeys were used to determine the virulence ofthe original samples of blood, as well as of the newly isolated cultures,
10rcutt, M. L., Y. Exp. Meg., 1924, xl, 43, 627.
Bauer, J. H., not yet published.
~Noguchi, H., and Battistini, T, S., Y. Exp. Meg., 1926, xtiii, 851.
4 Noguchi, H., ar. Exp. Meg., 1927, xlv, 175.

224

ETIOLOGY OF OROYA FEVER.

X

the tests being made by three different methods. (1) The effects oflocal inoculation of the original samples of blood were simultaneously
compared by intradermal inoculation of each specimen into separate
sites on the abdominal skin of the same monkey (the experiment wasdone in duplicate), previous experience having shown 5 that an experimental local lesion in Macacus rhesus remains confined to the site of
inoculation, and that several intradermal inoculations with different
materials may be made simultaneously on the same animal. (2) The
effect of local inoculation of each of the cultures isolated from the blood
TABLE

I.

Tests of Infectivity of Blood, Cases 1 to 6. Multiple lntradermal [noculallons,Dec. 29, 1926.
Case No.

M . rttes~ts IA

M . fltesu$ 2A

Blood cultures, Jan. 6, 1927.

I

-

I

Blood cultures, Jsn. 24, 1927.

]+ (1:100)[+ (1:10o)

was compared in the same animal with that of the inoculation ofpassage strains of known virulence. (3) Each strain was individuallytested on a monkey by both intradermal and intravenous inoculation.
The first two types of test have an advantage over the third in thatthe variations in the susceptibility of individual monkeys is not involved. The third method is the more usual one, but in testing alarge number of strains it is necessary to economize in monkeys, hence
the factor of individual variation may enter into consideration. The
results in this series of animals, however, were determined not onlyb y local reactions but also by blood cultures at appropriate intervals.
5 Noguchi, H., J. Exp. Med., 1927, xlv, 455.

HIDEY0 NOGUCHI225

For virulence tests, cultures grown for 8 days on leptospira medium
at 30°C. and those grown for 4 to 6 days on horse blood agar slants
and plates at the same temperature were pooled, the mixture comprising first, second, and third generation cultures of a given strain.
The original samples of blood were tested again simultaneously withthe cultures, a procedure which yeilded interesting results, inasmuch as
the blood, in contrast to the cultures, produced no cutaneous lesions.
The results of the various tests are outlined in the tables.
Table I summarizes the results of the direct pathogenicity testsof the blood of Cases 1 to 6 inclusive. No local lesions were induced,
TABLE II.
Paralld Infectivity Tests of Blood of Cases 2, 3, and 5, and of Cultures Obtained
from the Blood. Multiple Intradermal Inoculations, Feb. 7, 1927.
Case No.

M. rhesus 3A

Blood. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 kCulture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

++~Blood. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 ~Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

++++~Blood. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 kCulture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

++++though invasion of the blood by BartoneIla bacilliformis occurred, as
shown by the results of blood culture in both animals 26 days after
inoculation. The cultures from Cases 1, 4, and 6 were contaminated,
those from Cases 2, 3, and 5 yielded marked local lesions (Table II;
Fig. 31). Similar results were obtained with the specimens from
Cases 7 to 12, the original blood being non-vlrulent for the skin of themonkey, while the ~tfltures iaa three instances (Cases 9, 10, 11) gave
rise to typical verruga lesions. Parallel tests of the blood and thecultures in Cases 7 to 12 are recorded in Tables I I I and IV. Strains
7 to 12 were tested separately on different monkeys (Table V) withresults which agree with those of the multiple tests.
The experiments show that of nine strains tested, six possesseddefinite specific pathogenicity for Macacus rhesus; the other three226

ETIOLOGY

OF O R O Y A F E V E R .

X

were non-pathogenic, as shown by three separate tests. The three
non-virulent strains (7, 8, and 12) came from cases of benign miliaryor nodular verruga, in two of which the microscopic examination of
the blood had been negative for Bartonella; in Case I2 Bart~nella waspresent in the blood, though the red count was practically normal.
The six virulent strains, with the exception of Strain 10, came frompatients whose blood showed either microscopically detectable numTABLE

III.

Parallel Infectivity Tests of .Blood of Cases 7 to 12 and of Cultures Obtained fromthe )31ood. Multiple intradermal Inoculations, May 3, 1927.
Case No.

M. rhesus 4A

M. rhesus 5A

7~Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(Culture .........................................

8~Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~Culture ..........................................

9

I Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
lCulture .........................................

+++

+++

10

~Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
(Culture .........................................

+++

+++

11

~Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~Culture .........................................

++++

++++

++++

++++

12

m

Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture ....... ..................................

C o n t r o l (P. 5 strain, f r o m v e r r u g a nodule) . . . . . . . . . . . . . .

bers of Bartonella bacilliformis or marked anemia, or both. In threeof these cases there were no skin lesions, and the red counts were verylow (1,080,000 to 1,250,000). These represent the pure septicemic
form of Carrion's disease, without cutaneous involvement.
The fact that the viruIent strains came from the severe types ofCarrion's disease, and the non-virulent ones from benign verruga
seems to indicate that the highly fatal disease is caused b y strains
possessing a greater virulence. The first strain of Bartondla bacilli-

HIDEYO NOGUCHI

227

formis isolated, 8 which was obtained from the blood of a fatal case of
Carrion's disease, was found to produce in monkeys of average susceptibility the clinical picture of a human case of vcrruga, but in unusuallysusceptible animals, which are rather rare, it induced a fatal infection
similar to that of human Oroya fever. The strains from the severe
TABLE IV.

Parallel Infectivity Tests of the Original Blood, of Cultures on Leptospira Medium,and of Blood Broth Mixtures, Cases 7 to 12. Multiple Intradermal Inoculations, May 18, 1927.
Macacusrhesus6&
Case No.

Cultu~sOriginal blood
Bloodbroth

Leptospira medium

30t
7

37*

m

8

m

+++
++
+
+

9
10
11
12

TABLE V.

Individual Tests of Strain, 7 to 12. InlradermaI and Intravenous (2 co.)Inoculations, July 1, 1927.
Strain No.

Monkey No.

7

7A
8A
9A
10A
llA
12A

8

9
10
11
t2

Skin lesions
D

+
+++
++++

Blood cultures--(Aug. 8, 1927)+ (1:10)+ (1:100,000)

cases of the present series have not so far, in the small number ofmonkeys inoculated, reproduced the fatal Oroya fever, but they haveinvariably induced cutaneous lesions s'nnilarto those occurring inbenign human cases, while the strains isolated from the blood of
benign verruga have not proven sufficientlyvirulent to set up any
infection in the same monkeys.

228

ETIOLOGY

OF OROYA F E V E R .

X

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229

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230

ETIOLOGYOF OEOYAFEVER.

X

As has previously been suggested, the variety of types of human

Bartonella infection may be accounted for by variations in the susceptibility of different individuals or different tissues of the same
individual, or by variations in the virulence of the microorganism, orby both. The present experiments appear to emphasize more particularly the factor of variation in virulence of the parasite. According
to clinical observations, the skin lesions more usually follow the acutefebrile anemic stage (fiebre grave de Carrion, or Oroya fever); thelesions in these instances are evidently caused by a strain virulent
enough to have invaded the blood. Yet there are cases in which theinfection is chiefly confined to the cutaneous tissues, the anemia being
very mild, and strains isolated from such cases would presumably
be less virulent, as was found to be true in the present experiments.
In instances in which the severe bartonellemia supervenes in the courseof an apparently benign verruga, we may conclude that the strain
belonged to the virulent type, but that the individual was of theresistant type, and that the defenses of the resistant organism were
finally overcome by continued constitutional disturbances, due perhaps to the presence of the parasite in question, perhaps to some othercause. Phenomena of this sort are not uncommon in infections withother organisms which may give rise to septicemia following local
infection (streptococcus, staphylococcus, the tubercle bacillus).

Serologic Studies.
Suspensions of Bartonella bacilliformis which would be suitable for
agglutination tests are difficult to obtain, owing to the fact that themasses of organisms are well nigh impossible to disperse. Complement fixation tests proved easier to interpret.
Two samples of immune sera, obtained by inoculating rabbits intravenously on several occasions at appropriate intervals with live cultures of Strain S. A.8 were employed against a non-fixing dose of saline
suspensions of plate cultures (horse blood agar) of each strain of
Bartonella bacilliformis. The suspensions were heated at 60°C. tokill the microorganisms and the immune sera inactivated at 56°C.
All the strains gave complete fixation with the antiserum with the
exception of Strain 12, which gave partial fixation (30 per cent). The

moEYo ~ootTcm

231

results indicate that on the whole the strains belong to the same serologic group.
DISCUSSION.

Carrion's disease offered a singularly complex problem, because of
its two clinically dissimilar aspects, the grave fever of Carrion (Oroya
fever), and benign verruga. The presence of the endoglobular bodies
discovered by Barton* practically settled the etiology of t h e severecases, but the relation of Barton's bodies to the cutaneous lesions ofverruga remained obscure, since it had not been proven that the same
bodies were present in the blood or skin lesions of patients with themild cutaneous disease. Moreover, while the cutaneous tissues
affected were definitely infective, inducing similar skin lesions in monkeys, the parasites present in such large numbers in the blood ofpatients with malignant anemia, with or without skin lesions, appeared to be non-infective for monkeys, inducing neither skin lesions
nor systemic infection so far as could be determined. It has recently
been shown, however, that the apparently negative findings were notdue to the absence of Bartonella bacilliformis, but to the fact that,except in severe systemic infection, the parasite cannot be detected by
microscopic examination, its presence being revealed only by a suitableculture method. ~ The unsolved portion of the problem of the etiology
of Carrion's disease has been cleared up by the cultural procedure,
which permits the ready isolation of Bartonella bacilliformis and its
detection even when it is present in extremely small numbers.
The importance of the selection of the culture method cannot be overemphasized, since on the special features of the mediumused depended
the success of the entire investigation. Had blood agar been used forthe isolation experiments--and it is by far the best of the many othermedia tried--many strains would undoubtedly have been missed,
some because growth of the microorganism is not uniformly successful
on this medium, some because of loss of virulence due to the frequentsubcultuHng required with blood agar cultures. Once the parasite
had been obtained in culture on the semisolid leptospira medium, andits special properties studied, it became possible to discover the causes* Barton, A. L., Cr6n. meal., Lima, 1909, xxvi, 7.
Noguchi, H., J. Exp. Med., 1926, xliv, 697.

232

ETIOLOGY OF OROYA FEVER.

X

for the apparently conflicting clinical and experimental evidence.
Moreover, by special histological technique 8 it was shown that Bartonella bacilliformis is microscopically detectable in large numbers inhuman and experimental lesions, where it had previously escaped
detection.
The reason for the difference in pathogenicity between the endoglobular parasites, which are present in such large numbers inverruga maligna, and those existing in the skin lesions of verruga
benigna has not been brought to light. The fact that in the firstmentioned condition the microorganism is incapable of inducing inmonkeys anything more than a slight transitory blood invasion,
detectable only by blood culture, explains the negative results obtained by the Harvard Commission 9 with blood from severe Oroya
fever, results which seemed unmistakably to indicate a totally different
etiology for Oroya fever and verruga peruana.
SUM~fARY.

Through the cooperation of Dr. Sebastian Lorente, Director of the
National Department of Public Health of Peru, nine strains of Bartonella badlliformis have been isolated, by means of the semisolid
leptospira medium, from nine of twelve specimens of blood withdrawn
from cases of verruga and forwarded from Peru under conditions ofrefrigeration. The cultural titer of the Mood specimens immediately
after their arrival (2 weeks after withdrawal) varied from 1:10 to
1:100,000. Blood from the severe anemic type of the disease, in which
there was no eruption, had the highest titer. Blood agar slants yielded
irregular results, but some strains grew well on these media.
8 The technique is that used by Nicholson (J. Exp. Med., 1923, xxxvii, 221)
for the detection of Rickettsia in tissues. Fixation in Regaud's fluid (Arch.
Anat. Micr., 1910, xi, 296), which consists of 4 parts of 3 per cent potassium
bichromate and 1 part of commercial formalin, is followed by staining with
Giemsa's solution, according to the method recommended by Wolbach (J. Meal.
Research, 1919-20, xli, 1). 1.25 cc. of Giemsa's solution (Griibler's, for bacteria)
is diluted with 1.5 cc. of methyl alcohol and 50 cc. of distilled water to which
has been added 3 drops of 1 per cent sodium bicarbonate.
9 Strong, R. P., Tyzzer, E. E., Sellards, A. W., Brues, C. T., and Gastiaburu,
I. C., Report of first expedition to South America, 1913, Harvard School of
Tropical Medicine, Cambridge, 1915.

HIDEY0 NOGUCHI233

Morphologically the strains differed very little in fresh preparations
examined by dark-ground illumination. In stained preparations somestrains appeared coarser, others finer than the average. Special staining indicated that the flagella were characteristically unipolar andvaried in number from one to four, some strains showing distinctly
more wavy and heavier flagella than others. Young cultures grownon the surface of horse blood agar for 3 to 6 days show individuals withfairly sharp contours, short rods, often varying in thickness toward
one or both ends, being intermingled with smaller oval or coccoid
elements.- Some strains show a predominance of bacillary, some ofcoccobacillary forms. It is not known whether these features are
inherent or are due to conditions of growth, which, though identical,
may react differently upon different strains. Definiteness in outline
disappears with the age of the culture.
More striking variations are found in the virulence of the different
strains for the monkey (Macacus rhesus). Three of the nine strains
isolated proved to be non-pathogenic for the monkeys. All three ofthese were derived from cases of benign verruga. The remaining six
strains all gave rise to local lesions when intradermally inoculated andwere recovered in culture from the blood of the animals. So far,severe anemia has not developed in any of the monkeys.
It is significant that most of the severe cases yielded virulent strains,while some of the strains from benign verruga were non-pathogenic.
It appears highly probable that the severe form of Carrion's disease
is, in general, caused by a virulent strain, while the benign forms are
due to a strain of low virulence. On the other hand, a virulent strain
may cause benign verruga in unusually resistant persons and a weakstrain may give rise to severe blood infection in unduly susceptible
individuals. The form of Carrion's disease is probably determinedprimarily by the inherent virulence of the strain of t~artonella bacilliformis and is modified secondarily by individual predisposition in agiven case.
An interesting phenomenon brought out by the present investigation
was the failure of the nine human blood specimens to induce local
verruga in the same monkeys in which the corresponding cultures,
inoculated simultaneously at separate sites, gave rise to typical lesions.
Yet the original blood samples were shown by cultivation to have

234

ETIOLOGY

OF OROYAFEVER.

X

contained live bartonellas at the time they were inoculated, and blood
culture revealed the presence of the microorganisms in the blood ofmonkeys which showed no other signs of infection after inoculation
with the human blood. Whether this striking difference is merely aquantitative one or is due to some factor still unknown--such as, forexample, a biological phase of the microorganism--has not been
determined. The uniformly negative results of transmission experiments with blood by previous investigators is explained by an actual
inability of the blood to induce skin lesions and the lack, until now,of a reliable method of detecting Bartonella bacillijormis in the monkeys' blood.
The strains isolated showed similar serologic properties, as tested
by complement fixation.

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

PLATE 7.

Comparison of different strains of Bartonella bacilliformis.
Giemsa's stain.

X 1000.

Gram's stain, counterstained.

(Noguchi: Etiology of Oroya fever. X,)

T H E J O U R N A L OF E X P E R I M E N T A L M E D I C I N E V O L .

XLVII.

Comparison of different strains of Barlonella bacilliformis.
Giemsa's stain.

PLATE 8.

X 1000.

Gram's stain, counterstained.

(Noguchi: Etiology of Oroya fever. X.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. X L V I I .

PLATE 9.

Comparison of different strains of Bartonella bacilliformis.

× 2000.

Zettnow-Fontana stain.

(Noguchi: Etiology of Oroya fever. X.)

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

PLATE 10.

Lesions produced by cultures of (a) Strain 2, (b) Strain 3, (c) Strain 5, and (d) Strain
P. 5. 30 days after inoculation. M. rhesus 3A. Natural size.

Abnormal detached flagella. Dark field.

× 1000.

(Noguchi: Etiology of Oroya fever. X.)