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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
]~I. COMPARISON OF BARTONELLA BACILLI~'ORMISAND BARTONELLA
M~P~S. CULTrCATIONOF BACTERIIY~ ~YrRI-tYM, N. SP.
BY HIDEYO NOGUCHI, M.D.
(From lh¢ Laboratories of The Rockefeller Imtitule /or Medical Research.)
PLATES1t and 12.
(Received for publication, November 14, 1927.)
Mayer, ~ in 1921, discovered, in the red blood cells of a rat which was
suffering from severe anemia as a result of trypanosomiasis andchemotherapy, peculiar inclusion bodies; resembling Bartonella b ~ l i formis of Oroya fever, which he designated Bart~elta muris. Subsequently Landa -° reported that splenectomy of white rats brought
about a severe anemia which could be transferred to normal rats by
inoculating them with the blood or a suspension of liver tissue of thesplenectomized animals. The name "infectious anemia of the rat"
was introduced to describe the condition. Mayer and his collaborators8
suspected that the anemia resulting from extirpation of the spleen
might be due to Bartonella muris and succeeded in demonstrating theinclusion bodies in splenectomized rats. Their assumption was that
Bartonella muris is present in most rats, and that the invasion of theblood is made possible by the removal of a defense organ, the spIeen.
Because of the close analogy between Bartol~ella muris and Bartonella b~illiformis--their morphological similarity, their localizationin the erythrocytes, and their association with a severe form of anemia,
a comparative study has been made of these two bartonellas, thespecific subjects of inquiry being: (1) the percentage incidence ofappearance of Bartonella muris in the blood of splenectomized rats
and mice, (2) the result of splenectomy in animals other than ratst Mayer, M., Arch. Schi.ffs- u. Trope~J~yg., 1921, xxv, 150.
2Lauda, E., Arch. ges. Physiol., 1925, cclviii, 529.
s Mayer, M., Borchardt, W., and Kikuth, W., Klin. Woch., 1926, v, 559.
235

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ETIOLOGY OF OROYA FEVER.

XI

and mice, (3) the transmissibility of BartoneIla muris to normal rats,(4) the transmissibility of Bartonella muris to other animals, especiallysplenectomized animals, (5) the susceptibility to Bartonella baciUiformis of rats and other animals after splenectomy, (6) the comparative morphological features of Bartonella muris, •artonella bacilliformis,and Grahamella talpx, the latter an intracorpuscular parasite of moles
described by Graham-Smith,* Thomson,~ and Brumpt, 6 and (7) thecultivation of Bartonella muris.
Splenectomy was performed on fifty white rats, three wild rats,
three Chinese hamsters (Cricetulus griseus), ~ and nine white mice.
Bartonella muris appeared after splenectomy in the blood of all, insome numerously, in othei:s sparsely. Splenectomy always gives
rise to an increase in the number of blood platelets, some of whichshowed very much more distinct chromatin rods and granules thanthe platelets of normal animals. These loosely packed or partiallyliberated thrombocyte bodies are sometimes very easily confused with
Bartonella muris, especially when lying over or under the erythrocytes.
Splenectomy was carried out also in eight Macacus rhesus, two Javamonkeys, four rabbits, and four guinea pigs. No intracorpuscularelements resembling Bartonella muris or Bartonella baciIliformis appeared in the blood of any of these animals, nor did the animals show
any anemia comparable to that observed in the splenectomized ratsor mice.

Transmissibility of BartoneUa muris.
Repeated attempts have been made to reproduce the anemic condition and cause the appearance of Bartonella muris in the blood of
normal rats and mice by injection of blood or suspensions of livertissue (Lauda's method) from splenectomized animals, intravenously,
intraperitoneally, intradermally, or intratesficularly. Such experi* Graham-Smith, G. S., J. Ityg., 1905, v, 453.
6Thomson, ~. D., J. ttyg., 1906, vi, 574.
*Brumpt, E., JBull. Soc. Path. Exot., 1911, iv, 514.
TI am indebted for these animals (which were obtained primarily for otherexperiments) to Dr. Carl TenBroeck, who procured them for me in Peking, andto Dr. Amos Wong, who was so kind as to bring them to the United States in
his personal care.

HID~YO NOOUCHI

237

m e n t s h a v e always been negative in this respect. N o r has it beenpossible to t r a n s m i t the condition to s p l e n e c t o m i z e d monkeys, rabbits,
or guinea pigs.
I n some instances, however, a quite different condition was induced.
In Rats 54 and 55, inoculated intratesticularly with a suspension of the liverof Rat 40, which had shown a marked blood invasion by Bartone2la muris, thetesticles became acutely inflamed. Rat 54 was castrated on the 6th day of
disease. The condition persisted in Rat 55 for 10 days, but by the 14th day thetesticle had healed. Bar~one21amuris was not found in the blood of either ofthese animals. Histological study of the excised testicle of Rat 54 showed acuteinfiltration of polymorphonuclear leucocytes into the acini, together with edema,congestion, and focal necrosis. Rather coarse bacilli were present in the lesion,some having been taken up by the leucocytes. From the testicular tissue wasobtained a pure culture of a microorganism to be described later (Strain 54).
Two normal rats, 56 and 57, inoculated intraperitoneally and intratesticularly
with the suspension of testicular tissue of Rat 54, became very ill within 48 hours,
and in the blood of Rat 56, taken on the 3rd and 4th days, were found a few
intracorpuscular bodies suggestive of l~arione~la muris. The animal died before
the 5th day. Rat 57 showed similar elements in the erythrocytes on the 3rd
day but none thereafter. I t died of diarrhea in 10 days. There was only slightinduration of the testicles in these two animals.
A similar result was obtained in one of three normal rats inoculated withcitrated blood of splenectomized Rat 41 (B. muris -k q-). In this case (Rat 66)
a bilateral intratesticular inoculation had been made. The inflammation of thetesticles reached its maximum on the 5th day and receded during the 3 following days. The same microorganism as had been isolated from Rat 54 was obtained from the aspirated fluid (Strain 66). I11 the other two animals (Rats 64
and 65), in which the inoculation was unilateral, there was no induration. Examination of blood film.q of these three animals failed to reveal the presence of
B. muris. Three other normal animals inoculated intratesticularly and intraperitoneally with a suspension of the liver of Rat 41 showed neither testicular
induration nor invasion of the blood by B. muris.
There is a p p a r e n t l y present in the blood or liver of some splenectomized rats a microorganism capable of setting up acute orchitis innormal rats. T h e transient a p p e a r a n c e of intracorpuscular bodies inthe two r a t s inoculated with the testicular tissue of R a t 54 was far
f r o m convincing evidence of transmission o f / ~ . muris. T h e failureof m o s t of the a t t e m p t s to t r a n s m i t the organism shows a t least t h a t
regular transmission is not readily accomplished in A m e r i c a n rats.

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ETIOLOGY OF OROYA FEVER.

XI

Mayer, s in Hamburg, encountered a similar difficulty. It would,
indeed, seem unusual that a microorganism which is unable to invade
the blood until the spleen is removed should acquire the power toinfect the blood of a normal animal by transfer from the splenectomized one.
It has been found that the intraperitoneal deposition of a small
fragment of normal rat or mouse spleen, or the intraperitoneal injection of a saline suspension of the tissue, into splenectomized rats ormice will cause the rapid disappearance of BartoneUa muris from theblood. Hence B. muris is extremely sensitive to splenic substances,even when the spleen itself is not present.

Susceptibility to Bartonella baciUiformis of Splenectomized Animals.
Experiments to be reported elsewhere have shown that the spleen
has no important defensive function against the invasion of Bartonella
bacilliformis. While the blood culture fiters of splenectomized monkeys infected with Bartonella bacilliformis were usually higher thanthose of control animals of the same series, yet variations in blood titerare so common in monkeys infected with this microorganism that theycannot be taken as significant in this instance. Rabbits, guinea pigs,
and rats were not any less resistant to infection with Bartonella bacilliformis after splenectomy than are these animals normally2

Comparative Morphology of B. mutts, B. bacilliformis, and Grahamella
talpa~ in the Blood (Plate 11).
Comparison of blood films stained with Giemsa's solution shows

B. muris (Fig. 2) to be smaller than B. bacilliformis (Fig. 1).
There is also less variation in form in B. muris, which usually appears
as short rods or coccoid forms whose size varies within a very small
range. B. bacilliJormis, on the other hand, though often having an
oval or coccoid form similar to that of B. muris, frequently occurs inlong, slender rods, and Y or V forms are very common (Fig. 1).
The long slender forms usually have square ends, as though the ors Mayer, M., Borchardt, W., and Kikuth, W., Arch. Schiffs- u. Tropenhyg.,
1927, xxxi, 295.
9 Noguchi, H., J. Exp. Med., 1927, xiiii, 851.

~imr.Yo NOG~Icm

239

ganism had been cut transversely. Shorter rods may be bent at a
sharp angle at one or two places, showing that they are composed of
two or three individuals. These branching forms are not found in
preparations of B. muris, which resembles GrahameUa talp~emore thanit does B. baciUiformis, though differing from GrahameUain its mannerof distribution. I am indebted to Dr. A. C. Coles, of Bournemouth,
England, for Giemsa-stained preparations of the blood of two English
moles showing GrahameUa infection (Fig. 3). The grahamellas areoften found in such numbers in a single cell as to fill the stroma completely; B. muris is usually scattered among many cells, and many
individuals are found outside the cells. The ~reparations show twot)~pes of GrahameUa, one distinctly and uniformly thick, as comparedwith B. muris, and suggesting a bacterium, the other more delicate
and showing beaded formations. No branched forms or long threads
have been seen in the films of GrahameUa.
B. muris, B. bacilliformis, and G. talp~e have similar staining reactions with Giemsa's solution, but basic fuchsin, while giving sharp
definition of B. bacilliformis, fails to stain B. muris.
B. muris of the rat is indistinguishable from the similar elements
which appear in the blood of wild rats or Chinese hamsters after
splenectomy. The forms found in the corpuscles of splenectomized
mice are more granular and show beaded formations.

CultivatiOn of B. muris.
The etiologic r61e of B. muris in the anemia of splenectomized rats
is difficult to establish because of the fact that no animal has so far
been found which is susceptible to the organism under natural conditions, i.e., when the spleen is present. Once the spleen is removed,
B. rnuris appears in the blood, hence splenectomlzed animals cannot
be used for demonstration of the pathogenic effects of a microorganism
which has been isolated.
In the course of repeated attempts at cultivation, I have isolated
two different organisms, both of which resemble B, muris in morphology. The first (Strain 28 A, Figs. 7 and 8) came from the blood of asplenectomized rat and grew on leptospira medium as a grayish layer
at the surface. It did not grow on ordinary Culture media. It was

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ETIOLOGY OF OROYA FEVER.

XI

s u b s e q u e n t l y found t h a t this organism h a d the features of a m i n u t ediphtheroid. I t h a d no p a t h o g e n i c i t y w h a t e v e r for normal rats.
F r o m the blood of the same animal ( R a t 28) a n o t h e r minute, app a r e n t l y non-motile, G r a m - n e g a t i v e b a c t e r i u m was isolated on a
blood agar p l a t e (Strain 28 B, Figs. 4 to 6). This organism grows on
blood agar in v e r y m i n u t e colonies b u t not on leptospira m e d i u m oron o r d i n a r y culture media. On a blood a g a r slant it grows m o r ereadily, a n d after 3 or 4 d a y s a t 30 ° or 37°C. the dew-like discretesurface colonies gradually spread to a d i a m e t e r of 3 m m . or m o r e and,
when the surface is densely seeded, coalesce to a shiny, faintly bluishgray, m o i s t layer of growth. T h e condensation w a t e r becomes slightly
turbid. This organism is not an acid-producer like the first, b u t ishemolytic. Morphologically, especially when stained for a shortt i m e (20 minutes) w i t h G i e m s a ' s solution it a p p e a r s v e r y m u c h like
B. muris. T h e m o s t interesting feature of the organism is its ability
to set up a n acute orchitis in normal r a t s when intratesticularly
injected.
The first experiment was made on Rat 42, into which a 24 hour culture grownon blood slants at 30 ° and 37°C. was injected intraperitoneally and intratesticularly. B. rauris did not appear in the blood, but the testicle became indurated
within 6 days, when the animal was killed for transfer. A suspension of thetesticular tissue was used for cultivation experiments and for intraperitoneal
and intratesticular inoculation of two normal rats, 52 and 53. A pure cultureof the organism was isolated from the testicular tissue. No induration followed
the intratesticuiar injection of the tissue suspension, but in the blood of.Rat 52
a few forms resembling ]Bartondla muris were detected 8 days after the inoculation.
The virulence tests of Strain 28 B were repeated on two normal rats, 58 and 59,
the cultures being injected intratesticularly only. An acute orchitis developedin both animals, and they died 10 and 9 days after inoculation, respectively.
B. muris was not found in the blood at any time. From the testicles of Rat 59
pure cultures of the organism were recovered.
Pure cultures of Strain 28 B from Rat 42 were tested on two normal rats,
60 and 61, the injections being made directly into the testicles. Rat 60 reacted
more vigorously than Rat 61, the inflammation of the testicle reaching its maximum in 3 days and disappearing in 9 days, while Rat 51 showed only a slightinduration on the 3rd day. Bodies resembling "B. mur/s were found in verysmall numbers in the blood of Rat 61, but not in Rat 50. A saline suspensionof the testicular tissue of Rat 42, kept at 4°C. for 7 days, was injected into two
normal animals, 62 and 63, but in neither was there more than a very slightinduration of the testicles. B. muris was not found in the blood.

HIDEYONOGUCHI241

Strain 28 B proved, therefore, to be identical in pathogenicity andcultural properties with the strains isolated from Rats 54 and 66.
All three pathogenic strains are evidently the same microorganism.
The culture forms are small, measuring 0.4 to 0.8 ~ in length and 0.2
to 0.3 # in width, and appear very much like Bartonella muris. The
organisms found in the indurated testicular tissues were coarser
TABLE I.

Summary of Experiments witt, t]~eOrganism Cultlvated.
Sourceof culture

Strai

Blood of splenectomiz~ Rat 28

28

Non-pathogenic

Blood of splenectomized Rat 28

28

Induced testicular lesion in Rat 42. Culture recovered from testicular tissue
Suspension of testicular tissue induced
slight induration of testicles in Rats
62 and 63

Testicular tissue of Rat 42, inoculated with culture of Strain
28 B

28

No testicular lesions in Rats 52 and 53.
Few iutracorpuscular elements suggestive of B. mur/s seen in blood of Rat 52
Induration of testicles in Rats 58, 59, 60,
61. Culture recovered from Rat 59

Testicular tissue of Rat 54, inoculated with suspension offiver tissue of splenectomized
Rat 4054

Slight induration of testicles in Rats 56
and 57. Intrscorpusculsr elements insmall numbers in blood of beth

Testicular punctate of Rat 66, inocniated with citrated blood of
splenectomized Rat 41

66

Not tested

Resultof inoculationto.

(1 to 1.5 ~ by 0,3 to 0.4 g), took a deeper stain, and showed very little
resemblance to the delicate B. muris of the blood of splenectomized
rats.
Several normal rats, rabbits, and guinea pigs were injected intratesticularly with pooled cultures of Strains 28 B, 54, and 66 (fromRats 28, 42, 54, 59, and 66). The rats showed the usual marked
induration of the testicles, but the other animals reacted only slightly.
B. muris did not appear in the blood of any of the animals.

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ETIOLOGY OF OROYA F E V E R .

XIImmunization Experiments.
Several normal rats were inoculated intraperitoneally on several
occasions with live cultures of Strains 28 B, 65, and 66, and 9 to 10
days after the last injection the animals were splenectomized.
Following the splenectomy B. muris promptly appeared in the bloodof all, that is, previous active immunization with the microorganism
in question conferred no protection against the invasion of B. muris
on subsequent splenectomy.
An immune serum pepared in a rabbit by repeated intravenous
injections with live cultures of different strains of the pathogenic
microorganism failed to cause the disappearance of 23. muris from theblood of splenectomized rats or to prevent invasion of the blood by
B. muris thereafter when given intraperitoneally immediately before
splenectomy.
Mthoug'h neither active nor passive immunity could be induced inrats agalnst B. muris, by means of the microorganism under study,
this organism is not definitely proven not to be B. muris since according to recent observations of Mayer and his coworkers no immunity
develops in splenectomized rats as a result of previous infection, theorganisms appearing again in the blood of recovered individuals whenblood containing them is intracardially injected. In the case of
B. bacilliformis killed cultures do not constitute a potent vaccine, andan immune serum prepared in rabbits by repeated intravenous injections of live cultures of the organism confers little or no protection
against a virulent strain of the parasite, the incidence of takes among
vaccinated and control monkeys being practically the same.
The identity of the pathogenic microorganism cultivated fromsplenectomized rats cannot be established until suitable experimental
animals are found. In the meantime I propose for it the provisional
name, Bacterium murium.
Bacterium murium was not isolated from the blood of 20 normal
rats. Its invasion of the blood of splenectomized animals wasperhaps due to the removal of the spleen.
SUMMAkY.
BartoneUa muris appeared in the blood of all white rats, wild rats,
Chinese hamsters, and mice, from which the spleen was removed, but

BIDEYO NOGUCm

243

did not appear in that of splenectomized monkeys, rabbits, or guineapigs.
I t has not been possible to transmit B. muris to normal rats, monkeys, rabbits, or guinea pigs, by intraperitoneal, intradermal, or intravenous injection of blood containing B. muris from splenectomizedrats.
In two instances an acute orchitis was induced in normal rats byinjection directly into the testicle of blood or saline suspensions of
the liver of splenectomized rats. The intracorpuscular elements
occasionally found in the blood of some of the animals Could not be
definitely identified as B. muris or as having appeared as a result of
the inocul~ation. The acute orchitis of rats was transferable to normal rats in series.
From the testicular tissue, as well as directly from the blood of asplenectomized rat, there was isolated in pure culture a microorganism
which induced in the testicles of normal rats an acute orchitis suchas resulted from inoculation of the blood or liver suspensions of
splenectomized rats. While a few inclusions were found in the erythrocytes of some of the animals, their number was so small and theiroccurrence so infrequent that they could not be definitely identified
as B. murls.
In morphoIogical features the cultural forms of the microorganism
isolated resemble B. murls. The organism found in the testicular tissues, however, is considerably coarser than B. muris and takes a deeper
stain.
Immunological study failed to settle the question of the relation
between B. muris and the cultivated organism, which is provisionallycalled Bacterium murium.
BartoneUa muris, BartoneUa bacilliformis, and GrahameUa talpae
have characteristic individual morphological features.

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

PLATE 11

(Noguchi: Etiologyof Oroya fever. XL)

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

PLATE 12.

(Noguchi: Etiology of Oroya fever. XI.)