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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
vn.

P,.ESPONSE

SXlN OF MACACUS :RHESUS AND

ANTHROPOID APES TO INOCULATION WITH BARTONELLABACILLIFORMIS.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, November 8, 1926.)

In animals experimentally infected with Bartonella bacilliformis, 1-4
spontaneous involvement of the skin is far less frequent, and rarely
so extensive, as in human verruga. In man the organism probably
enters the body through the skin, and, after a preliminary multiplication elsewhere--perhaps in the lymphatic glands--invades the skinfrom within, giving rise to a persistent endothelial hyperplasia. In
animals, when Bartonella bacilliformis is introduced locally, a lesion
arises which may in time assume dimensions as great as the humanverruga nodule. But notwithstanding the persistence of the lesion
for many months, and the fact that the surrounding skin is frequently
bathed in blood resulting from mechanical injury to the lesion, no new
nodules develop. Hence it becomes of interest to determine whether
the resistance of the skin in monkeys is due to an anergic condition,such as exists in syphilis, or to inability of the parasite to invade theintact skin.
Two other questions presented themselves in this connection, (1)whether it would be possible to induce verruga formation by injuring
an area of normal skin in an animal having active skin nodules elsewhere and carrying Bartonella bacilliformis in the blood, and (2)whether autoinoculation would be successful in actively infected
1 Noguchi, H., and Battistini, T. S., J. Exp. Med., 1926, xliii, 851.
2 Noguchi, H., J. Exp. Med., 1926, xliv, 697.
3 Noguchi, H., J. Exp. Med., 1926, xliv, 715.
* Noguchi, H., ]. Exp. Med., 1926, xliv, 729.

455

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

VII

animals, i.e., whether skin i m m u n i t y develops during the course of
infection, a n d if so, at w h a t time.

Resistance of the Intact Skin to Bartonella bacilliformis.
T h e question whether Bartonella bacilliformis is capable of inducing
lesions when applied to the surface of the intact skin is one of practicalimportance. T o test the point, we applied to intact areas of skin whatever m a t e r i a l was being inoculated elsewhere. T h e infectious m a t e r i a l
was rubbed on two separate areas, each several cm. square, and usuallynear the sites of inoculation b y scarification or i n t r a d e r m a l injection.
N o cutaneous lesions were ever induced b y this method, though lesions
i n v a r i a b l y developed at the sites of the i n t r a d e r m a l injections and in
m a n y instances also on the areas inoculated b y scarification.
Although unusually extensive lesions (verruga mular) are often
produced b y application of infectious m a t e r i a l to scarified areas of theskin, there h a v e been m a n y instances in which this m o d e of inoculation failed, while typical lesions (verruga nodular) always arose at
the sites of i n t r a d e r m a l injection. H e n c e it is desirable always to useb o t h m e t h o d s of inoculation.
Chimpanzee, 8 Pan leucoprymnus, about 4 years old. .Tan. 19, 1926, abdominal
skin on the right side was shaved and two areas, each about 3 X 4 cm. square,
were scarified and smeared with a saline suspension of the skin nodule* of M.
rhesus 3. At the same time two adjacent unscarified areas were rubbed with thesame suspension. Mar. i, 1926, pinkish, raised indurated lesions had become
noticeable on both scarified areas, l~artonella bacilliformis was demonstrated in
the lesions, both in sections and by culture. No lesions developed on the areas
which were not scarified before being smeared with the suspension.
Ourang-utan, 3 Pongo pyg~emus, about 3 years old. Apr. 17, 1926, abdominal
skin shaved and an area (2 × 3 cm.) on the right lower quadrant scarified andrubbed with a piece of nodule from the eyebrow of M. rhesus 23. An unscarified
area on the upper quadrant was vigorously rubbed with the same material.
May 20, 1926, pinkish, raised, linear lesions, rather pale, have been present on the
scarified area for some time. Bartonella bacilliformis was demonstrated in thelesions both by culture and in sections. No lesions developed on the unscarified
smeared area.
M. rhesus 18.2 Feb. 15, 1926, right eyebrow and skin of right abdomen shaved
and scarified and a saline suspension of the nodule of M. rhesus 5 applied. Adja* Nodules were removed under ether anesthesia.

mD•Yo NOO~Cm

457

cent unscarified areas were rubbed with the same material. Extensive nodules ofdeep red color developed on the scarified area of the eyebrow within a few weeks
and numerous punctiform red miliary nodules on the scarified abdominal skin.
The abdominal lesions disappeared within a month, the nodules on the eyebrow
remained until the middle of June, when they became small, pale, and fibrous.
Bartonella bacilliformis was demonstrated in the lesions both by culture and insections. No lesions developed on the unscarified smeared areas.
M. rhesus 23.5 Mar. 9, 1926, left eyebrow shaved and a scarified area smearedwith a piece of the nodule from the eyebrow of M. rhesus 18. An adjacent unscarified area was vigorously rubbed with the same piece of tissue. Within 2 to
3 weeks large protruding lesions had appeared on the scarified area, and these
progressed to considerable size during the following weeks. Bartonella bacilliformis was found in the sections of the lesion. No lesion appeared outside of the
scarified area.

Relation of Injuries to Localization of Lesions.
W h e t h e r or not mechanical factors--friction, exposure to minor
i n j u r y - - a r e involved in the spread of the skin lesions in h u m a n verruga
is not known. In animals these devices appear to have no influence,as shown b y repeated failure to induce localized lesions in infectedanimals b y scarifying the skin or introducing foreign substances (agar,
culture medium) intradermally during the height of the infection, when
Bartonella badlliformis was demonstrable in the blood. In one unusually susceptible animal, M. rhesus 25, 2 in which spontaneous miliary
nodules developed, scarification of the abdominal skin had no influenceon the course of the general eruption. Similar a t t e m p t s to induce
ncdale formation failed also in the chimpanzee, the ourang-utan, andin M. rhesus 18, 2 which had at the sites of scarification on the eyebrow
one of the most extensive local lesions (verruga mular) observed in
the course of the experimental work with Bartonella bacilliformis.
I t is evident that in experimental animals Bartonella bacilliformls
cannot be made to localize in an area of skin injured either b y scarification or b y intradermal inoculation of foreign substances, though
present in the circulating blood and in the lymph channels, as well as
in the skin lesions. Injuries of the skin are not equivalent to the deposition of c o n c e n t r a t e d infectious material on the scarified skin or in
the cutaneous tissues.
5 Noguchi, H., J. Exp. Med., 1927, xlv, 437.

458

ETIOLOGY OF OROYA FEVER. VII

A utoinoculability and Superinf ection.
In a disease like verruga peruana, which persists over a long period,
there must be a steady process of self-infection. The manner in which
the cutaneous lesions spread has not been definitely determined, but
it is certain that the skin must retain its susceptibility during periods ofremission and relapse. The skin of Macacus rhesus and that of theapes is relatively so insusceptible to infection with Bartonella bacilliformis that spontaneous eruptions (verruga miliar) seldom occur,
but it has been possible to induce well marked nodular lesions (verruga nodular) in actively infected animals by intradermal inoculation of suspensions of nodular tissue either from the same or fromanother animal. Such nodules never attained the size of the primarylesions, and they usually began to recede earlier. In no instance wasit possible to induce any lesions on scarified areas during the course of
infection, though the materials employed induced typical lesions incontrol animals.
In infections with the strain of Bartonella bacilliformis derived from
Oroya fever, the existence of active lesions considerably reduced
the susceptibility of the skin to subsequent inoculation. Moreover,once the animals were free from Bartonella bacilliformis, they werecompletely refractory to reinoculation, hence the reduction in susceptibility during the course of illness is due to a partial immunity, not toa state of anergy.
On the other hand, in infections with the strain of Bartonella bacilliformis from a case of verruga, 6 there have been exceptional instancesin which a preexisting infection did not prevent the development of
the most severe type of local infection after reinoculation with thesame (verruga) strain. M. rhesus 33 and M. rhesus 34 showed thistype of reaction.

M. rhesus 33 was inoculated on Apr. 7, 1926, with the saline suspension of ahuman verruga nodule (Case P 5). Bloodtaken on Apr. 16 and again on May
27 yielded (in 1 : 10 dilution) cultures of Bartonella bacilliformis.
On June 3 the animal was inoculated intradermaUyand by scarificationwith thesuspension of a nodule from the eyebrow of M. rhesus 41 (secondgeneration of thesame strain). On June 24 the lesionshad already de'celopedat the sites of inoculation, and by July 1 they were very large and active.
s Noguchi, H., J. Exp. Med., 1927, xlv, 175.

HIDE¥O NOOUCHI

459

M. rhesus 34 was inoculated on Apr. 10, 1926, with the saline suspension of thenoduleof Case P 5. Cultureswas obtained from diluted (1 : 10) blood on April 28,
May 12, and May 27. By June 29 a large nodule had developed on the tail.
On June 3 the animal was inoculated intradermally and by scarificationwith thesuspension of the eyebrow nodule of M. rhesus 41. Bartonella badlliformis wascultivated from a 1 : 100 dilution of blood taken on June 20. Large lesions had
appeared at the sites of inoculation at this time, and by July 1 the lesions had
become unusually extensive.
As these instances show, it is not possible to make any generalstatement with regard to the partial immunity observed in mostanimals when passing through a protracted course of infection with
Bartonella bacilliformis. Immunity may develop so slowly in someanimals as not to interfere with the course of a superinfection.

Pathogenesis of the Skin Lesions.
The verruga-like skin lesion prod.uced by BartoneUa bacilliformis
in anthropoid apes and M. rhesus is a slowly progressing infectious
angioendothelioma and owes its origin to a delicate reciprocal relation
between the endothelial cell and the microorganism. Bartonella
bacilliformis finds within the cytoplasm of endothelial cells most favorable conditions for its multiplicative phase, and once lodged there
multiplies steadily, though perhaps rather slowly, eliciting certain
reactions on the part of the infected cells. Since these parasitizedcells remain alive for a long time and undergo active mitosis, it isclear that the parasite furnishes a mild type of stimulus which leads
to continuous proliferation of the cells. The lesion thus started continues to grow until local as well as general immunity intervenes toterminate the peculiar association of cells and parasite.
The evolution of the verrnga lesion in experimental animals may be
divided into (1) the incubation period, (2) the initial stage, (3) the
mature vascular stage, and (4) the regression. These divisions areof course arbitrary, one stage passing into the next imperceptibly, yeteach at its height has its special feature.
Incubation Period.--No instance has been observed in which thelesion was macroscopically recognizable within less than 10 days after
inoculation. Usually a trace of induration can be detected after 10
to 14 days, though in some cases the incubation period is as long as 3
weeks.

460

ETIOLOGY OP OROYA F E V E R .

VIIInitial Stage.--When the nodular lesion, which becomes noticeable
as a slightly raised area, 1 to 2 mm. in diameter, has reached a sizeof 4 to 6 ram. and protrudes 2 to 3 ram. beyond the surrounding normalskin, the color becomes slightly pinkish, and edema of the surrounding
tissues becomes evident. The nodule is now firm and well definedto the touch and can be easily separated from the overlying skin andthe loose connective tissue in which it is embedded. The cut section
is grayish pink, edematous, but not soft, and microscopically the
nodule is seen to consist of masses of endothelial cells, placed close
together, lying among capillary vessels and connective tissue fibers.
A small number of lymphocytes and polymorphonuclear leucocytes
are seen in the interstices of the cuffs, but not within the nodule itself.
The endothelial walls of the capillaries are thickened, and the luminaof the blood vessels narrowed. Practically every endothelial cell in
the nodule contains Bartonella bacilliformis, some cells being packed
with clumps of organisms. The endothelial cells lining the capillaries
are often filled with similar masses of microorganisms. The histological picture at this stage is very simple.
Mature and Vascular Stage.--The interval from the preceding sta~eto this one varies from a few to 10 to 14 days and persists for 10 days
to 2 weeks.
The increase in size of the nodule may be gradual or even relatively
rapid until the lesion measures 8 to 10 mm. or more in diameter. The
color first becomes bright, then deep, red. The overlying skin isshiny, and the epidermis scaly in places. The nodule is still firm andis sharply demarcated from the surrounding tissues. The color may
change to a dark bluish red, suggesting that of a cherry. This type oflesion, especially when deep seated and less vascular on the surface, ischaracteristic of the form of human lesion known as verruga nodular.
Occasionally the overlying skin yields to the increasing pressure fromwithin, and the lesion becomes pedunculated.
The lesions found on the scarified lines of the skin are seldom round;on the contrary, they are usually irregular at the surface, and the massresembles a raspberry in form and color, being made up of a number ofsmall, translucent, red, drupelet-like nodules. The entire region
adjacent to the lesion is edematous. The lesion is devoid of an epidermic layer, hence it bleeds readily on slight friction. This type of lesion
corresponds to that termed in man verruga mular.

~mEYoNOOUCm461

In a few animals spontaneous nodules have appeared on the skinat sites remote from those inoculated; these correspond to the generaleruption in human cases known as verruga miliar. Such lesions
have always been small, none becoming larger than 1 to 2 ram. indiameter, but their clinical course is similar to that of the nodules
induced by intradermal inoculation or by scarification.
On microscopic examination, nodules or lesions removed during
the mature and vascular stage present a somewhat more complex picture than those of the initial stage. The large number of capillary
vessels intersecting dense masses of closely packed endothelial cells andthe increase of fibrous connective tissue irregularly penetrating thenodular structure are striking features of the lesions. Numerous fibroblasts and fibrils lie between islands of angioblasts, the appearance
somewhat suggesting that of a fibrosarcoma. In places there is hyperplasia of the epidermis, and some large detached masses of epithelioid
cells find their way deep into the nodular zone, a circumstance which
complicates the interpretation of the ce]lular elements composing thenodule. Some of these invading epithelioid cells show hydropic
degeneration. Degenerating endothelial cells and numerous erythrocytes are present in some places and are taken up by migrating macrophages, which also engulf the dead polymorphonuclear leucocytes
simultaneously present in these areas. Along the interstitial spaces
penetrated by connective tissue fibers, plasma cells and mast cells
are sometimes rather numerous. The lumina of the capillary vessels
are often occluded or compressed by proliferating endothelial cells.
Mitotic figures are common. The skin may be adherent.
The microorganisms are more uniformly present in the endothelial
cells along the periphery of the nodule than among the cells in the older
foci. None except endothelial cells contain Bartonella bacilliformis in
the cytoplasm, and no extracellular localization of the parasites canbe detected.
When a secondary bacterial invasion has taken place, there arefound, needless to say, an enormous number of polymorphonuclear
leucocytes loaded with the contaminating bacilli or cocci. At times
these bacteria-carrying leucocytes are taken up by endothelial cells
and give rise to a confusing appearance, as though the secondary
invaders were the primary cause of the hyperplasia.

462

ETIOLOGY OF OROYA F E V E R .

VIIRegression.--The time of beginning of the regressive process depends
on the individual animal's resistance to the infection, and the processof regression is intimately associated with the acquired immunity, local
or constitutional. Early regression, before full development of thelesion, has been rather frequent in the experimental animals. Oncethe nodule has fully developed, however, considerable time is requiredfor its complete resolution, 4 to 5 months in some instances. The firstindication of retrogression is the gradual loss of the deep red color. As
the color fades the size of the nodule begins to diminish, the paling anddiminution continuing until finally only a tiny colorless wart remains.
When the nodule ruptures spontaneously at the beginning of the periodof regression, the whole mass sloughs and the wound heals rapidly withscar formation. The result is quite different, however, when the
nodule is injured, or partially removed by surgical operation during
its developing stages; in this instance the lesion acquires renewed
vigor and spreads far beyond its previous limits in the form of averruga mular; it becomes malignant.
The histological appearances of the lesions, as just presented, aresimilar to those observed by the Harvard Commission 7 in human andexperimental verruga tissues, of which they carefully studied a large
number. The eosin-staining inclusions so constantly encounteredin the endothelial cells, both in human and monkey nodules, and particularly emphasized by the Commission, are of considerable significance. These reddish granules, which may have been identical withthose described by Mayer, Rocha-Lirna, and Werner 8as Chlamydozoa,
in all probability represent intracellular forms of Bartonella bacilliformis, somewhat less distinctly stained than in our preparations.
The possibility of indistinct staining is one which cannot be ruled out
in connection with this difficultly staining m!croorganism.
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.
s Mayer, M., Rocha-Lima, H., and Werner, H., Mi~nch.reed. Woch., 1913, lx,
739.

HIDE¥O NOGUCHI463

SUMMARY.

Bartonella bacilliformis failed to induce lesions when merely rubbed
on the surface of the intact skin of a chimpanzee, an ourang-utan, andnumerous Macacus rhesus monkeys, although when applied to thescarified skin of the same animals it gave rise to extensive lesions.
Application of infectious material to the scarified skin did notalways induce verruga lesions, but intradermal inoculation almost
invariably gave rise to nodule formation.
The localization of Bartonella bacilliformis in the skin is not, inexperimental animals, determined by mechanical factors, since scarification of the skin or intradermal injection of foreign substances inmonkeys infected with Bartonella bacilliformis does not give rise toverruga formation.
The degree of susceptibility of the skin tissues appears to be considerably diminished during the course of experimental infection with
Bartonella bacilliformis. Inoculation of the scarified skin of infectedanimals gave uniformly negative results, and intradermal inoculationinduced only a mild local reaction. In a few exceptional instances,
however, of animals previously infected with the strain of Bartonella
bacilliformis derived from a human verruga nodule, reinoculation
with the same strain gave rise to unusually marked reactions.
The evolution of the skin lesion induced in experimental animals byBartonella bacilliformis may be divided into four stages, the period of
incubation, the initial stage, the mature and vascular stage, and theregression. In the initial stage the lesion is a pure angioendothelioma,
but in the stage of full development the histological picture is complicated by connective tissue proliferation and occasionally also by penetration of epidermis into the lesion. The demonstration of Bartonella
bacilliformis in the endothelial cells distinguishes the lesion from others
which simulate it.
The cutaneous lesions known as verruga nodular, verruga mular,
and verruga miliar have been reproduced in monkeys.