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E T I O L O G Y OF OROYA F E V E R .
VI. PATHOLOGICALCHANGESOBSERVED IN ANIMALSEXPERIMENTALLY
INFECTED WITH BARTONELLA BACILLIFORMIS.
THE DISTRIBUTION OF THE PARASITES IN THE TISSUES.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
PLATES I I T O 14.
(Received for publication, October 20, 1926.)
Experimental infection with Bartonella bacilliformis in young
Macacus rhesus monkeys varies in severity according to the susceptibility of the individual animals 1.*,s and is of three general types:(1) severe, fatal affection characterized by extreme anemia, similar to
Oroya fever in man, (2) relative benign condition, in which there ismoderate anemia or none at all, accompanied by localized skin lesions
resembling those of h u m a n verruga, (3) mild systemic disease, involving the lymphatic glands and blood.
The pathological changes found in human organs and tissues after death from
Bartonella bacilliformis infection have been described by Odriozola,* Arce,5Hercelles, 6 and by Strong and his coworkers,r The'characteristic effects are seenin the liver, spleen, lymphatic glands, and bone marrow. Hypertrophy of theliver is almost invariable,a, 5and the spleen is frequently enlarged, 4' 7though it may
1 Noguchi, H., and Battistini, T. S., J. Exp. Med., 1926, xliii, 851.
2 Noguchi, H., J. Exp. Med., 1926, xliv, 697.
s Noguchi, H., J. Exp. Med., 1926, xliv, 715.
40driozola, E., La maladie de Carrion, Paris, 1896.
5Arce, J.,An. Facul. Med. Lima, 1918, i, No. 1, p. 21; No. 2, p. 130; No. 3,
p. 240; No. 4, p. 24.
s Hercelles, 0., An. Facul. Med. Lima, 1918, i, 10.
7 Strong, R. P., Tyzzer, E. E., Sellards, A. W., Brues, C. T., and Gastiaburfi,
J. C., Report of first expedition to South America, 1913, Harvard School of
Tropical Medicine, Cambridge, 1915.
437

438

ETIOLOGY OF OROYA FEVER.

VI

be of normal size or less. 5 Infarcts are commonly found in the spleen and sometimes also in the liver.6' z Strong and his coworkers note particularly the areas ofdegeneration in the liver, beginning about the hepatic veins, apparently due tothe activity of the parasite and suggesting the presence of a toxin in the circulating
blood. The lymphatic glands are enlarged everywhere4' 5, ~ and frequently
edematous. The hypertrophy is most marked in the mesenteric lymph nodes. 4
The marrow of the long bones is red or mottled with red patches, 4' 7 and distinctly
softer than normal. Microscopically there is evidence of phagocytosis by endothelial leucocytes of red cells and polymorphonuelear leucocytes, also increased production of normoblasts. ~ Extensive phagocytosis by endothelial cells is found also
to occur in the spleen, while the lymphatic glands often contain many large swollen
endothelial cells in which the bacilliform parasites are present in large numbers, z
Similar intracellular parasites may be found ~ in the blood vessels near ulcerated
areas in the intestinal wall. The parasite has a marked predilection for the
reticulo-endothelial system and sets up an active and persistent proliferation ofendothelial cells in the tissues or organs which it invades.
The characteristic nodular lesions caused by Bartonella bacilliformls haveoccasionally been found to occur in the muscles and viscera as well as on the
mucous membranes.4' 5, s, 9 Campod6nico, in 1895,1° reported the finding at
autopsy, in a young child who succumbed to severe anemia during the course ofverruga, of tiny red nodules in all the tissues of the body.
I n the early experimental work with Bartonella bacilliformis only
the milder types of infection were observed, a n d it was not until thethird a n d fourth generations in m o n k e y s t h a t it was possible to induce, b y massive inoculations of passage strains, a fatal disease accompanied b y extreme anemia c o m p a r a b l e with O r o y a fever of m a n . F i v eanimals succumbed to the infection, and, as in the case of the h u m a ndisease, the conspicuous changes were found in the liver, spleen, bonemarrow, a n d l y m p h a t i c glands. I t was desirable, therefore, not only
to c o m p a r e the findings with those occurring in h u m a n material, b u t
also to relate the histological changes in a particular tissue with thepresence or absence of the parasite. I n order b e t t e r to accomplish
this purpose a n u m b e r of infected ianimals were sacrificed either a t
the height of disease, as evidenced b y extensive local lesions a n d ahigh titer of the parasite in the blood, or during the period of convaless Odriozola, E., Cr6n. m~d., Lima, 1914, xxxi, 157.
9 Hercelles, O., Cr6n. mSd., Lima, 1914, xxxi, 67.
10 Campod6nico, E., CrSn. m3d., Lima, 1895, xii, 43.

HIDEYO NOGUCHI

439

cence, when the blood findings were negative and the local lesions had
subsided.
The important fact should be emphasized that tuberculosis was notencountered among the animals of the present series, notwithstanding
special search was made for B. tuberculosis because of the possibilityof confusion from this source.
Although microscopic examination of all materials--blood, organs, or tissues-was always made, the cultural method was found to be more reliable for determining the presence of Bartonella bacilliformis. Moreover, secondary infection
is readily detected by this method. About 0.1 cc. of each of several dilutions (from1:10 up to 1:1,000,000) of blood or saline suspension of tissue is introduced into
leptospira medium, and the tubes are kept constantly at 25°C. for 4 to 5 weeksand
examinedperiodically. Growthis often detected only after 2 to 3 weeks.
Films were always made of blood and impression smears of spleen, bone marrow, lymph glands, and liver. They were dried in air, fixed in methyl alcohol for
5 to 10 minutes, and stained with Giemsa's solution for 30 to 60 minutes.
Tissues were fixedin Regaud's fluid, and the sections stained with hematoxylin
and eosin for histological study and with Giemsa's solution for detection ofBarlonella bacilliformis.
The fourteen monkeys (Macacus rhesus) serving as material for thepresent study will be considered in three groups: (1) five animals whichdied of experimental infection with Bartonella bacilliformis; (2) fiveanimals which showed signs of infection, locally and constitutionally,
when sacrificed; (3) four animals which had shown signs of infection
but were apparently in convalescence when sacrificed.
In the first group, three showed marked and two extreme anemia
during the latter part of the disease or at the time of death. In
Monkey 25 the red blood cells numbered 1,164,000 per c.mm. and thehemoglobin (Sahli) was 25 per cent; in Monkey 37 the red cells
numbered 1,176,000 and the hemoglobin was 15 pe r cent. In theseanimals a terminal secondary bacterial infection intervened 12 and96 hours before death.
There was moderate anemia in some of the animals of Group 2 but
none in those of Group 3.
In Group 1 (fatal cases) the clinical picture was that of Oroya fever,and the pathological changes likewise proved to be similar to those440

ETIOLOGY 01~ OROYA FEVER.

VI

observed in the h u m a n infection. In Group 2 (non-fatal cases), themanifestations were characteristic of h u m a n verruga.

Pathological Findings in Fatal Infections with Bartonella bacilliformis.
T h e following outline is a composite picture of the characteristicchanges noted. T h e findings in individual instances and the relationof the cultural to the pathological findings are recorded in Table I
and in the protocols.
T h e duration of illness was 56 days in M o n k e y 17, 36 days in Monkey 25, 25 days in M o n k e y 30, 29 days in M o n k e y 35, and 41 daysin M o n k e y 37.

External Appearances.
The skin and mucous membranes have a striking waxy yellowish appearance,
and the numerous swollen lymphatic glands in the axillary and inguinal regions
stand out prominently beneath the stretched skin. Pale nodules are found at theoriginal sites of inoculation on eyebrows and abdominal skin. In an exceptional
instance (Monkey 25) there were also numerous spontaneously occurring nodules
on various parts of the body.

Thorax.
Lungs.--Pale yellow, normally crepitant, smooth. Congestion or edema atbase in some instances. Bronchial lymph nodes greatly enlarged. Pleurae
anemic; small amount of effusion present.
Heart.--Myocardium flabby, pale, contains partially fluid pale red blood andoccasionally a small clot. No petechi~e present. The pericardium contains someclear fluid. In one instance (Monkey 25) an acute pericarditis due to secondary
invasion was present.
Abdomen.
Liver.--Pale, flabby in consistency, frequently somewhat enlarged; often shows
yellowish mottling. Areas of necrosis and fatty degeneration are often
recognizable.
Spleen.--Dark bluish red, enlarged, rather firm in consistency. Follicles indistinct. Scattered minute grayish red areas (infarctions) are present in all instances in varying numbers.
Kidneys.--Pale, with yellowish gray tint.
Adrenals.--Pale yellow.
Pancreas.--Normal in appearance.
Stomach.--Usually empty, mucosa pale.
Small Intestine.--Pale, serosa studded with numerous dark bluish swollen
lymph nodes. Mucosa pale. Peyer's patches slightly hypertrophied.

H I D E Y O NOGUCHI

441

Large [ntestlne.--Similar in appearance to small intestine, swollen lymph nodes
on serosa giving it a striking aspect. No ulceration of the mucosa.
Bladder.--Normal in appearance.
Ovaries, Uterus, Testicles.--Pale, but normal.
Bone Marrow (Femur).--Dark grayish red.
Lymphatic Gla~ts.--Entire lymphatic system hypertrophied and congested toan extraordinary degree. In the inguinal and axillary regions two or three swollen
glands sometimes unite to form an irregular mass, 2 to 3 cm. in longest diameter,
pink to deep red in color, and edematous. In the abdominal cavity the swollen
lymph nodes are sometimes dark bluish, sometimes bluish red, and by contrast
with the pale intestinal wall present a spectacular picture. They range in size
from 2 to 6 ram. and are attached so firmly to the intestinal wall that they appear
like verrucous nodules.
Nervous System.--The brain and spinal cord and their coverings are anemic but
otherwise appear normal.
Microscopic Pathology.
Liver.--The normal pattern of cell arrangement is more or less disturbed. The
hepatic cells have lost their sharp outline, stain poorly, and are vacuolated. The
cells around the hepatic veins are necrotic (Fig. 1). Here and there, on the borders of the necrotic areas, between the liver cells, are engorged macrophages, which
often contain erythrocytes, polymorphonuclear leucocytes, and erythroblasts.
Around the portal veins are several layers of proliferated endothelial cells. In
some specimens yellowish or brownish pigment in granules or small masses isfound within the endothelial phagocytes or in endothelial cells lining the sinusoids.
The degree of necrotic change around the hepatic veins is variable in the different
animals. In some areas the liver ceils are clear and homogeneous, in otherscoarsely granular. A few endothelial cells containing Bartonella were found after
prolonged search in two of the five livers (those of Monkeys 17 and 30).
Spleen.--(Figs. 7, 8, 23.) Many infarctions are found, the periphery showing
invasion of leucocytes. Many of the veins are occluded, owing probably toendothelial hyperplasia. The splenic nodules are reduced in size and are often
scattered through the proliferating endothelial cells. Engorged macrophages containing erythrocytes are quite abundant. Yellowish brown pigment is present,
some in the endothelial leucocytes, some between the splenic cells.
A number of endothelial ceils containing masses of elements resembling
Bartonella bacilliformis were found (Figs. 16, 25), but none of the swollen
endothelial cells containing rods and granules such as were seen in humanmaterials by Strong and his coworkers.
Lymphatic Glands.--The principal changes in these glands are the increase in
the number of endothelial cells and the presence of numerous engorged macrophages. In the mesenteric nodes a large amount of greenish pigment is found in
the phagocytic endothelial cells of the sinuses. Bartonella bacilliformis has been
observed in a few endothelial cells in Monkeys 29 and 30 (Fig. 27).

442

ETIOLOGYO]~ O R O Y A

FEVER.

VI

Bone Marrow (Femur).--Numerous macrophages containing erythrocytes,
polymorphonuclear leucocytes, and cell d6bris are present, and there are large
numbers of normoblasts in some specimens (Monkeys 17, 25, 30). Some cells,
probably endothelial, containing elements resembling Bartonella bacilliformis
have been found in film preparations (Figs. 15, 26).
The changes are strikingly like those described as occurring in tissues from fatal cases of Oroya fever, the differences being in degree
rather than in kind. For example, the changes in the liver and spleen
in monkeys are decidedly less pronounced than those seen in suchhuman materials as are at m y disposal for comparison, while the reaction in the lymphatic glands is in reverse degree, the more active
phagocytosis being observed in monkeys. The origin of the minutenecrotic areas in the spleen may be sought in the obstruction of capillain lumina by proliferating endothelial cells.
As in the skin lesions, so also in the spleen and lymph glands, theprogressive and persistent proliferation of endothelial cells is usuallyassociated with the presence of Bartonella bacilliformis in considerable numbers, as detected by cultures. The extensive zonal necrosis
around the veins of the liver, which is less extensively invaded by theparasite, would appear, as suggested by Strong and his coworkers, tobe due to the presence of a toxin in the circulating blood.

Pathological Findings in Animals Sacrificed during the Period of Active
Infection.
The five animals of Group 2 all showed signs of active infection at
the time when they were killed by etherization. The period from
inoculation to the time of killing was 22 days in Monkey 4, 68 days in
Monkey 5, 53 days in Monkey 23, 24 days in Monkey 24, and 28 daysin Monkey 29. None of the animals showed severe anemia, although
in Monkey 24 there was moderate diminution both in red blood cells
and hemoglobin (erythrocytes 3,432,000, hemoglobin 55 per cent).
Bartonella bacilliformis had been cultivated from the blood of Monkey
23 in a dilution of 1:100,000 16 days after inoculation, but it could
not be recovered from undiluted blood 15 days later nor at any
subsequent time.
The spleen and lymphatic glands were usually affected, and also
the bone marrow as far as studied; the liver was less frequently in-

~mEYo ~ o a v c m

443

volved. Except in Monkey 23, of which special mention will be madelater, the other organs were always normal in appearance.
The spleen was always more or less enlarged and rather firm; in one
instance the surface was rather irregular (Monkey 5). In one animal(Monkey 23) there were numerous grayish brown areas of ififarction,
1 to 1.5 ram. in diameter, scattered over the organ, which was a dark
bluish red (Fig. 23). The lymphatic glands were in all cases swollen and congested and approached in size those of the fatally infectedanimals. The bone marrow was active in three animals studied (Monkeys 5, 23, and 24). The liver was affected to a similar degree in two
animals (Monkeys 23 and 24); in the other three it was apparently
normal. Bartonella bacilliformis was seen in a few of the endothelial
cells in the liver of Monkey 24. The microscopical findings in the animals of Group 2 were identical with those in the fatally infected animals except that the changes were less pronounced in some respects.
An unusually interesting observation was made in the case ofMonkey 23. On the pinkish surface of the lobules of the lungs were
noticed a dozen pale, grayish, semitransparent, round nodules, measuring 1 to 2 ram. in diameter, firm, and sharply demarcated from theadjacent normal lung tissue (Fig. 22). Microscopically (Fig. 11),they consisted of numerous large mononuclear cells, many of themvacuolated and filled with dark granular pigment. The capillaries inthis area, which were few in number, showed endothelial thickening.
Several engorged macrophages and cell d~bris were also present. Ina few of the endothelial cells Bartonella bacilliformis was found (Figs.
14, 24).

Pathological Cganges in Animals Sacrificed during Convalescence.
Four animals were sacrificed when apparently in the course ofconvalescence from a mild infection. None showed fever or local
lesions, though the lymphatic glands were still swollen in all instances.
The erythrocyte count ranged from 5,472,000 to 6,584,000, and thehemoglobin was 80 to 90 per cent. The animals appeared to be inexcellent condition when sacrificed 58 days (Monkey 7), 49 days
(Monkey 8), 40 days (Monkey 10), and 30 days (Monkey 13) after
inoculation.

~4~

ETIOLOGY

OF OROYAFEVER.

Vl

The changes in these animals, when still evident, were characteristic. The spleen was somewhat enlarged in three instances and in two
showed definite endothelial hyperplasia; in one (Monkey 13) there wasa marked increase in fibrous tissue; in another (Monkey 7) the samechanges v~ere noted as had been found in the spleens of the actively
infected animals. The liver was macroscopically normal in all fouranimals. The lymph nodes were active in two (Monkeys 7 and 8)
but quiescent in the others. The bone marrow was normal in Monkeys
8, 10, and 13, and active in Monkey 7.
The findings suggested that Monkeys 7 and 8 were still infected andthat Monkeys 10 and 13 had become free from infection. Cultural
studies confirmed this inference, as will appear later.
D I S T R I B U T I O N OF BARTONELLA BACILLI_FORMIS I N THE TISSUES I N THE
EXPERIMENTAL INFECTION.

In Oroya fever Bartonelta badlZiformis persists in the blood until thepatient's death. Whether or not various organs and tissues also
contain the living parasite has not been determined. The pronounced
changes found in certain organs have been assumed to be due
either to invasion by the parasite or to the action of a toxin circulating in the blood. Cultural determination of the distribution of theorganism in the tissues of experimental animals was, therefore, of
special usefulness in establishing a relation between the pathologicalprocesses and the presence of the parasite. The results of cultural
studies and of examination for pathological changes are recorded inparallel columns in Table I.
As the analysis shows, Bartonella bacilliformis was recovered fromblood, liver, spleen, lymphatic glands, bone marrow, and local lesions
in all instances of fatal infection, except when cultures were lost
through contamination. The parasite was not so generally distributed in animals sacrificed during the course of an apparently nonfatal infection, though the local lesions and the changes in the spleen
were rather severe. In Monkey 23 the parasite had disappeared fromthe blood, owing probably to the development of a blood immunity,
but persisted in the lymphatic glands and spleen, while in Monkey 29
the blood and nodular tissue contained it but the organs were all free

445

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446

ETIOLOGY OF OROYA FEVER.

VI

Summary of Patkologlca~
M.No.
rhesus

17

25

30

35

Liver
Inoculation Mar. 15, 1926. Died after 57
days. Fever still present. Nodules active. Anemia marked (r.b.¢. 3,112,000,
Hb. 50 per cent). Blood culture + at
autopsy. Nodule culture + at autopsy

Pale, somewhat enlarged. Structure disturbed, cells along central zones poorly
stained. Perivascular proliferation moderate. Greenish pigment present. B.
baciUiformis found in a few endotheliaiJ.
cells. Culture contaminated

Inoculation Mar. 23, 1926. Died after 36
days. Course practically afebrile. Local lesions extensive, partly generalized.
Anemia very severe (r.b.c. 1,624,000, Hb.
25 per cent). Blood (1:100,000) culture+ 12 hrs. before death. Culture of
nodules taken at autopsy +Pale, soft, mottled, perhaps enlarged.
Structure fairly well preserved but pronounced central necrosis throughout.
Numerous phagocytes, polymorphonudears, and pigments present. Hepatic
cells vacuolated. Culture contaminate~

Inoculation Mar. 31, 1926. Died after 25
days. Fever present. Nodules small
but active. Blood (1:10,000 dilution)
culture + 3 days before death. Anemia
moderate (r.b.c. 3,920,000, Hb. 60 percent). Blood and nodules taken at
autopsy yielded cultures

Pale, appears fatty. Normal structure lost,
cells stain poorly, vacuolated and dissociated. Endothelial hyperplasia; som~
cells contain B. bacilliformis. Many
phagocytes in sinusoids. Culture +Inoculation Apr. 12, 1926. Died after 29
days. Fever still present. Nodules
small but active. Anemia slight (r.b.c.
4,776,000, Hb. 80 per cent). Blood(1: 100) culture + 10 days before death.
Blood and nodules taken at autopsy
yielded cultures

Probably paler than normal. General structure disturbed. Marked dissociation andvacuolation of cells. Invasion of sinusoids by phagocytes. Proliferation ofendothelial cells. Culture +447

HIDEYO NOGUCHI:and Cultural Findings.
SpleenLymphatic glands

Bone marrow

Rema~s~,glarged, rather firm. Follicles reduced. Endothelial hyperplasia.
Activephagocytosis. Fibrin
masses and pigments present. B.
bacilliforrnisfound. Culture positive

All lymph nodes
much swollen andcongested. Endothelial hyperplasia
and active phagocytosis throughout. Culture +Dark grayish red.
Veryactivephagocytosis.
Numerous normoblasts
andpolymorphonuclear leucocytes
present.
Culture +B. bacilliformis~lightly enlarged, soft. Follicles
smaller, outlines of many lost.
Numerous perifollicular necroticfoci. Marked endothelial hyperplasia. Active phagocytosis of~degenerated polymorphonuclears
and erythrocytes. Many normoblasts present. Culture contaminatedMarkedswelling.
throughout.
Mesenteric nodes
appear like scattered or clusteredpeas but bluish incolor. Pronounced
proliferationofendothelial cells
and active phagocytosis. Culture
contaminatedDark grayish red.
Active phagocytosis. Polymorphonuclears
andnormoblasts present.
CulturecontaminatedTerminal bacterial
infection (pericarditis) intervened after heart
puncture 24 hrs.
before death

Enlarged. Follicles thinned anddiffuse in places. Endothelial
elements increased around vessels; small loci with fibrinous
exudate and cell d6bris. CUlture -[-Generalswelling. Grayish red. AcEndothelial hyperi
tive phagocyplasia,
activetosis.
Polymorphonudears
phagocytosis,
greenish pigments.
and normoblasts
B. bacilliformis
present.
Culfound in a few
ture +
endothelial cens.
Culture +B.

)ark and soft. Follicles ill defined,
pervaded by increased endothelial
cells. Active phagocytosis. Culture +Swelling and congestion throughout. Endothelial
hyperplasia.
Phagocytosis.
Culture W

B.

Grayish red. Active phagocytosis. Culture+

found in all tissues studied

bacilliformis
found in all tissues studied

bacitliformis
found in all tissues studied

448

ETIOLOGY

OF OROYAFEVER.

VI

Summary of Pathological.,M. r]w~u$
No.

37

23

24

Liver
Inoculation Apr. 17, 1926. Died after 41
days. Fever still present. Small nodule
onleg. Blood (1:1,000) + 96hrs. before
death; r.b.c. 1,664,000, Hb. 25 per cent.
Autopsy blood contaminated; anemia extreme (r.b.e. 1,176,000, Hb. 15 per cent).
Nodules (autopsy) +Pale and soft. General structure much disturbed. Cells ~tain very poorly, necrotic
along central zones. Small masses ofpolymorphonuclear leucocytes scatteredin sinusoids, where many loaded phagocytes are present. Endothelial .proliferation marked. Culture contaminated

Inoculation Dec. 8, 1925. Killed after 22
days. Fever present. Nodules active.
Blood culture at autopsy + . Nodules at
autopsy +Apparently normal.

No culture made

Inoculation Dec. 11, 1925. Killed after 68
days. Had become afebrfle. Nodules
large and active. R.b.c. 4,856,000, Hb.
85 per cent. Blood culture and nodules(autopsy) +Apparently normal.

No culture made

Inoculation Mar. 9, 1926.. Killed after 53
days. Fever present. Nodules active,
extensive. Blood + in 1:100~000, 40
days previously~ -- 22 days previously
and at autopsy. Nodules (autopsy) -F

General structure irregular; some ill staine~foci. Numerous
polymorphonudears.,Moderate endothelial hyperplasia. Culture contaminated

Inoculation Mar. 19, 1926. Killed after 24
days. Fever present. Nodules active.
Anemia moderate (r.b.c. 3,432,000, Hb.
55 per cent). Blood (1:10) and nodules
at autopsy +Pale, mottled. Moderate degree of central
necrosis, uniform structure lost in places..
Endothelial proliferation around vessels.
B. bacilliformis found in a few endothelial,
cells. Culture +449

HIDEYO N O G U C H I

and Cultural Findings.
Remarks

SpleenLymphatic glands

Dark and firm. Only a few follicles preserved. Necrotic areas(infarctions)
showing
activephagocytosis found. Increase inendothelial cells. Culture contaminated

Swollen and congested. Endothelial
hyperplasia. Active phagocytosis.
Polymorphonuclear leucocytes
present. Culture
contaminatedGrayish red. Ac- Terminal
secondaryinfection
tive phagocytosis and increaseby strain of B.
paratyphosus A
inpolymor48 hrs. before
phonuclear leucocytes.
Culdeathture contaminatedS~omewhat swollen and firm. Considerable increase in endothelial
elements. No culture madeEnlarged and congested
everywhere.
Definiteendothelial hyperplasia; active phagocytosls;
pigments. No culture madeNot examined

Enlarged, granular surface. Follicles ill defined in many places. A
few necrotic areas. Active phagoeytosis. Culture +Moderately swollen
andcongested.
Phagocytosis active. Hyperplasia
of endothelial cells.
Culture @Grayish red. Active phagocytosis.
Normoblasts present.
Culture W

S~mewhat enlarged, shows numerous brown patches of infarction.
Many perifollicular necrotic areas.
Active phagocytosis. Pigments
present.
Endothelial hyperplasia. Culture +red.
Greatly swollen and Grayish
Phagocytosis accongested. IIypertive.
Manyplasia of endothepolymorphonulial
elements.
clear leucocytes.
Very active phagoCulture +cytosis. Culture

Bone marrow

Pure infection with

B. bacilliformls

B.

bacilliformis
found in all tissues studied except
liver.
Animal moderately resistantNote absence of B.

bacilliformis

inblood and persistence in othertissues+"~hlarged, bluish red. Many foUides diffuse, interspaced by proliferated endothelial cells. Active phagocytosis. Many normoblasts. Culture +Swollen and congested. Endothelial
hyperplasia
and phagocytosis.
Culture +bacilliformis
Grayish red. Ac- B.
found in all tistive phagocysues
studied.
tosis. NumerAnimalquite
ous polymorphosusceptible
nuclearsandnormoblastspresent.
Culture +450

ETIOLOGY O F O R O Y A

FEVER.

VI

Summary of PathologicalM . rl~s=s
No.

29

10

13

Liver
Inoculation Mar. 30, 1926. Killed after 28 Mottled. Microscopically appears normal.
days. Fever had been present for 2 wks.
Culture -Nodules very active. Blood (1:1,000) +day before autopsy. Anemia noticeable.
R.b.c. 4,704,000, Hb. 70 per cent. Bloodand nodules at autopsy +Inoculation Dec. 21, 1925. Killed after 58
days. Fever had subsided 10 days previously. Nodule removed 30 days previously + . Blood q- 8 days previously.
Blood taken at autopsy + . R.b.c.
5,472,000, Hb. 80 per centAppears normal.

Culture contaminated

Inoculation Dec. 30, 1925. Killed after 49
days. Fever had subsided 7 days previously. Nodules absent. Blood -]- 14days previously, -- at autopsy. R.b.c.
5,496,000, Hb. 80 per centAppears normal.

Culture --Inoculation Jan. 8, 1926. Killed after 40
days. Fever had subsided 14 days previously, nodules 3 wks. previously.
Blood (1:100) -b 30 days previously, -at autopsy. R.b.c. 6,584,000, tIb. 65 per
centAppears normal.

Culture --Inoculation Jan. 19, 1926. Killed after 30
days. Fever had subsided 10 days previously. No nodules. Blood ~ 19 days
previously, -- 8 days previously, -- at
autopsy. R.b.c. 6,104,000, Hb. 90 per

Appears normal. Perivascular infiltratiog
present. Culture --centfrom it. In the latter instance the organism had apparently not yet
invaded the organs.
Of the four animals in which fever and local lesions had been present
but had subsided, two (Monkeys 7 and 8) proved to be still carrying
Bartonella bacilliformis in one tissue or another. Monkey 8 is especially interesting, as the latent infection was demonstrable only in thelymphatic glands. The other two animals (Monkeys 10 and 13) had

HIDEYO NOGUCHI

451

and Cultural Findings.
SpleenLymphatic glands

Bone m a r r o w

Remarks

gnlarged, bluish red. Follicles appear to be of normal size andstructure. Moderate endothelial hyperplasia. Numerous normoblasts. Culture -Swollen and congested. Hyperplasia
of endothelial elements, some phagocytosis.
Culture +Not examined

Liver and spleen
had probably not
yet been invaded.
Animal rather
resistantEnlarged, follicles prominent. On- Moderate swelling.
Slight endothelial
ly slight hyperplasia of endothelial ceils microscopically. Folliproliferation.
cles apparently normal. Culture
Culture ++Appears normal. Culture --Grayish red. No B.
bacilliformis
found in mostsections. Cultissues studied:ture +
evidenceofchronicity of infection

Marked swelling and Grayish yellow.
congestion. HyApparently normal.
Culperplasia of endothelial ceils. Phaturegocytosis active.
Culture +B.

bacilliformis

found only in
lymph
nodes.
Animalapparentiy convalescing

Probably enlarged. Microscopically normal. Culture --Definitely enlarged, Grayish yellow. Had
recoveredbut pale. Generalfrom infection
Normal.
Culmildnessofhyperplasia. Culture - which was probtureably due to resistance of animal~Stightly enlarged (?); surface uneven, firm. Definite increase infibrous tissues. Hyperplasia ofendothelial cells. Culture --fromConsiderable swell- Grayish yellow. Recovered
mild infection.
Apparently noring and congesind.
CultureHigh resistancetion.
Hyperplasia. Cultureof animal notedmcompletely recovered, as indicated by the histological findings andp r o v e n b y t h e n e g a t i v e r e s u l t s of c u l t u r a l e x p e r i m e n t s .
B r i e f p r o t o c o l s of a l l t h e a n i m a l s of t h e p r e s e n t series a r e r e c o r d e dabove.
SUMMARY.

T h e p a t h o l o g i c a l c h a n g e s o b s e r v e d in t h e o r g a n s in Macacus rhesus
m o n k e y s w h i c h h a v e s u c c u m b e d to s e v e r e i n f e c t i o n w i t h Bartonella

452

ETIOLOGY OF OROYA FEVER.

VI

bacilliformis are similar to those found in human organs in persons
dying of Oroya fever.
The characteristic changes in the liver are the zonal necrosis of thecells around the hepatic veins, involving active macrophagocytosis of
invading polymorphonuclear leucocytes in the necrotic areas, and amarked endothelial hyperplasia in the sinusoids or around the portal
veins. In some instances there is fatty infiltration of hepatic cells.
Ill the spleen persistent hyperplasia of the endothelial cells of thecapillaries leads to the formation of minute foci of infarction, owing to
occlusion of the lumina. The follicles are dispersed or reduced, andthere is an active macrophagocytosis of cellular d6bris, polymorphonuclear leucocytes, and erythrocytes. In some specimens an increasein normoblasts is noted. Pigment is sometimes present.
The lymphatic system shows general progressive endothelial hyperplasia, with active invasion of macrophages which contain polymorphonuclear leucocytes, erythrocytes, and greenish or dark pigments.
In the bone marrow there is increased activity of macrophagocytes.
Numerous normoblasts are found in some instances.
In one monkey, sacrificed during the course of injection, small, verrugalike nodules were found in the lungs and spleen.
Bartonella bacilliformis has been detected microscopically, thoughin small numbers, in all tissues showing histological changes. Parallelcultural determinations of the presence of Bartonella bacilliformis in
the blood, liver, spleen, lymphatic glands, bone marrow, and local lesions established the relationship between the pathological conditions
and the presence of the parasite. The organism seems to persist
longest in the lymphatic glands. Cultural methods offer a simple andconclusive means for the determination of the presence or absence of
the infecting organism.
EXPLANATION OF PLATES.
PLATE 11.
FIC. 1. Experimental Oroya fever. Section of liver from M. rhesus 25, showing
a rather characteristic zonal necrosis of the cells around the hepatic veins.
Giemsa's stain. X 50.
FIc. 2. The same section, X 182.
FIG. 3. Human Oroya fever. Section of Hver from the case (S. A. 15) fromHIDEYO NOGUCHI

453

which the strain of Bartondla bacilliformis employed in these investigations was
isolated. The necrotic changes of the liver cells are more diffuse than in theexperimental infection, but there is a definite tendency to a central type. Giemsa's
stain. X 50.
FIG. 4. The same section, X 18~.
FIG. 5. Human Oroya fever. Section of liver from the collection of the Harvard
School of Tropical Medicine. Courtesy of Professor R. P. Strong. This patient
was free from any verruga lesions and was regarded by the Harvard Commission
as a pure case of Oroya fever. The section shows the characteristic central necrosis. Note the striking resemblance between this preparation and that of themonkey liver in Fig. 1. × 50.
FIG. 6. The same section, X 182.
PLATE 12.
FIG. 7. Experimental Oroya fever. Section of spleen from M. rhesus 23. A
splenic follicle in an infarction zone, showing a perifoUicular necrosis and the invasion of the follicle by proliferating endothelial cells, endothelial phagocytes, andpolymorphonuclear leucocytes. Giemsa's stain. × 182.
FIG. 8. Experimental Oroya fever. Section of spleen from M'. rhesus 25.
An area of infarction where the splenic nodule has practically disappeared.
Giemsa's stain. × 182.
FIG. 9. Human Oroya fever. Section of spleen, for comparison. A splenicfollicle within the infarction, showing a general disorganization, necrosis, andphagocytic invasion. Giemsa's stain. × 182.
FIG. 10. Human Oroya fever. Section of spleen from another case. The
structure of spleen is greatly disturbed, shows general endothelial hyperplasia,
and the presence of macrophagocytes and polymorphonuclear leucocytes. Notethe striking resemblance between these lesions and those shown in Figs. 7 and 8.
Giemsa's stain. × 182.
F~G. 11. Section of one of the nodules in the lung of M. rhesus 23. Giemsa's
stain. × 50.
FIG. 12. The same section, × 182, showing the proliferation of endothelial
cells within the nodule.
PLATE 13.
FIG. 13. Bartonella 3acilliformis, colonies from a blood agar slant grown for 6
days 25°C. Smear preparation stained with Giemsa's solution for 1 hour, showing
the pleomorphism of the organism. Inserted here for comparison with the intracellular forms. × 1,000.
FIG. 14. Film preparation from a nodule in the lung of M. rhesus 23, showing
irregular masses of Bartondla 3acilliformis within the cytoplasm of an endothelialcell. Giemsa's stain. >( 1,000.
FIG. 15. Film preparation from the bone marrow (femur) of M'. rhesus 23,

454

ETIOLOGY OF OROYA FEVER.

VI

showing Bartonel~a bacilliformis within one of the endothelial cells. Giemsa's
stain. X 1,000.
FIG. 16. Film preparation from the spleen of M. rhesus 23, showing a cell(endothelial?) containing several small masses (Bartonella bacilliform,is?).
Giemsa's stain. × 1,000.
FIG. 17. Section of an experimental skin nodule produced in M. rhesus with astrain of Bartonella bacilliformis from verruga, n showing a dense mass of theparasites within an endothelial cell. Giemsa's stain. X 1,000.
FIG. 18. Section of an experimental subcutaneous lesion produced in the
chimpanzee 3 with the strain of Bartonella bacilliformis from Oroya fever, showing
irregularly scattered masses of Bartonella bacilliformis. Giemsa's stain. X 1,000.
FIG. 19. Section of a skin lesion experimentally produced in an ourang-utan, 3
showing a small mass of Bartonella baciUiformis (below) and a dense mass of
granules, probably of a mast cell (above). Giemsa's stain. X 1,000.
FIG. 20. Human Oroya fever. Section of lymphatic gland, showing two swollen
endothelial cells, loaded with minute granules, projecting into the lumen of acapillary vessel. Giemsa's stain. X 1,O00. From Case S. A. 15.
FIG. 21. Human Oroya fever. Section of lymphatic gland, showing theswollen endothelial cells lining a capillary vessel in an oblique section. Theseceils contain the very minute granules first described by the Harvard Commission
as an intracellular phase in the life cycle of Bartonella bacilliformis. (Courtesyof Professor Strong.) Giemsa's stain. × 1,000.
PLATE 14.

FIG. 22. The lungs of M. rhesus 23, showing the pale grayish nodules on both
lungs. They measured 2 to 3 ram. in diameter. In the middle, near the trachea,
is a grayish lymph node of the size of a pea. Natural size.
FIG. 23. The spleen of the same monkey, showing numerous infarctions.
Natural size.
FIG. 24. Bartonella baciIliformis in an endothelial cell in a film preparationfrom one of the nodules in the lung of M. rhesus 23. Giemsa's stain. X 1,750.
FIG. 25. t~artonella bacilli/ormis in an endothelial cell. Film preparation fromthe spleen of M. rhesus 23. Giemsa's stain. X 1,750.
FIG. 26. Bartonella bacilli/ormis (?) in an endothelial cell of the bone marrow(femur) of M. rhesus 24. Film preparation. Giemsa's stain. X 1,750.
FIG. 27. Bartonella bacillifor~nis in an endothelial cell of the lymph gland of
M. rhesus 29. Section. Giemsa's stain. X 1,750.
ll Noguchi, H., J. Exp. Med., 1927, xlv, 175.

PLATE 11.

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 V .

Liver, M. rhesus 25.

X 50.

Liver, M. rhesus 25.

X 182.

Liver, human Oroya fever.

X 50.

Liver, human Oroya fever.

X 182.

Liver, human Oroya fever.

X 50.

Liver, human Oroya fever.

X 182.

(Noguehi: Etiology of Oroya fever. VI.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLV.

PLATE 12.

(Noguchi: Etiology of Oroya fever. VI.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLV.

PLATE 13.

(Noguch;.: Etiology of Oroya fever. VI.~

THF_. JOURNAL OF EXPERIMF_NTAL MEDICINE VOL. XLV.

PLATE 14,

(Noguchi: Etiology of Oroya fever. ¥I.}