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Journal of Experimental Medicine

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ETIOLOGY OF OROYA F E V E R
XVI. Vv.RI~UGAIN ~Em DoG AND TEE DONXE~
BY HIDEYO NOGUCI-II, M.D., HENRY R. MULLER, M.D., EVELYN B.
TILDEN, PH.D., ANDJOSEPH R. TYLER(From the Laboratories of The Rockefeller Institute for Medical Research)
PLATES21 TO23
(Received for publication, July l, 1929)
In the older literature on verruga 1,2,8,4 are found statements thatin endemic centers the disease may sometimes occur spontaneously indomestic animals, particularly horses, mules, dogs, and pigs. That
distinct differences in the appearance of the nodular lesions existed,
or were thought to exist, among these animals, is apparent from thefact that they were used as criteria for describing certain types oflesions in man; for example, the deep-seated, bulky lesions were, andstill are, called "mular" or "mulaire," whereas the superficial ones
were sometimes referred to as "verrugas de caballo" (verruga ofhorses). The variation in appearance of the nodules seems to havebeen accepted by tradition, since careful descriptions of verruga inanimals cannot be found, and actual records of experimental inoculations in the closely related species of horses, mules, and donkeys areapparently very rare. The experiment of Ribeyro, Mackehenie, andArce,5 in 1913, with a donkey, is the only one of the kind on record.
The animal was inoculated by scarification of the skin at the inner
canthus of the eye, and on the nose, with the "pulp" of a verruganodule obtained from a patient. After 21 days the skin at the sitesof inoculation showed papules 3 to 4 ram. high, which increased onlyvery slightly in size during the next 6 days. Then they began toregress and after I I days had entirely disappeared. The reaction
seems to have been a very mild local one.
In their attempts to find an animal susceptible to Bartonella bacilliformis, Noguchi and Battistini ~ tried ringtail, rhesus, Java, and greenmonkeys, dogs, rabbits, mice, rats, and guinea pigs, and found that455

456ETIOLOGYOF

OROYA F E V E R .

XVIresults were definite o n l y in t h e rhesus m o n k e y . L a t e r , e x p e r i m e n t swere s t a r t e d (Noguchi) in dogs, horses, a n d d o n k e y s , t h e results ofw h i c h are r e c o r d e d below. I n t h e d o g mild v e r r u g a was induced, int h e horse a n d in b u r r o s t h e results were n e g a t i v e so far as c o n c e r n e dt h e p r o d u c t i o n of t r u e v e r r u g o u s skin lesions; in t h e horse a n d in oneb u r r o t h e r e was i n d u r a t i o n a t t h e sites of inoculation, w h i c h lasted 3t o 4 weeks, b u t Bartonella bacilliformis w a s n o t r e c o v e r e d f r o mt h e lesions. F u r t h e r experiments, w i t h d o n k e y s , h a v e been m o r e
successful.
Dog 1, female, inoculated March 23, 1926, intradermally, on eyebrows andabdomen, with (a) a mixture of 4-weeks old cultures, 1st and 2nd generations,grown on blood agar slants and leptospira medium, from the blood of M. rhesus
14, 7 a chimpanzee, r and M. rhesus 18, 8 and (b) a saline suspension of nodular
tissue excised* from M. rhesus 18. 2 cc. of the culture mixture were injected
intravenously. Small nodules developed after 8 days at the sites of culture
inoculation, but none at the sites inoculated with nodule suspension. Four days
later one of the nodules had reached a diameter of 0.5 cm. and was excised.*
Bartonella bacilliformis was recovered in culture from a suspension of the tissue.
Blood cultures made at this time were negative.
Microscopic examination of nodular tissue: In the spaces among the thick collagen fibers of the dermis there is a proliferation of endothelial cells. This process
is chiefly diffuse, but small loci also are found. There is also a considerable polynuclear exudate and a moderately extensive plasma cell exudate. Except forthe latter findings, the lesion is histologically similar to the lesions produced inmonkeys by Bartonella bacilliformis.
Colt, about 3 years old. Inoculated March 30, 1926, intradermally, at two sites
on the left flank, with mixed cultures (from blood agar slants and semisolid
leptospira medium) of Bartonella bacilliformis. There was no change in theanimal's temperature, which was normally 100.4°F. (38°C.), and blood culture
was negative 8 days after inoculation. Mter 20 days there was definite induration
at the sites of inoculation, which disappeared within the next two weeks. A
second inoculation was made at 4 sites on the neck, on April 22, with the material
inoculated on that day into Donkey 1. After 4 to 6 days there was marked induration at the two sites of culture inoculation, but none at the sites inoculated withnodular tissue and blood, and there was no fever.
Donkey 1. Inoculated April 22, 1926, at 4 sites on the left flank and 3 on theleft side of the neck, with (a) 15-day culture from blood agar slant, 5th generationfrom M. rhesus 7, s (b) 14-day culture on leptospira medium, 1st generation fromM. rhesus 29, 9 and (c) saline suspension of nodular tissue from M. rhesus 18, s* All operations were performed under ether anesthesia.

H. NOGUCHI, H. R. MULLER, E. B. TILDEN AND J. R. TYLER

457

and (d) citrated blood from M. rhesus 25. s 8 cc. of the mixture were injected into
the jugular vein. There was no change in the animal's temperature, which
remained at about 100°F., and blood culture was negative 4 days after inoculation.
At this time there was marked edema along the neck and flank and at otherpoints remote from the sites of inoculation. Mter 8 days there was definite
induration at the inoculated sites, and one of the "nodules" was removed for
culture and section. Bartonella bacilliformis was not recovered from the suspension of tissue, nor were the organisms seen in the sections. Histologically, however, the section has the appearance of that from a very early verruga lesion.
Several minute foci of endothelial cell proliferation are present in the deep portionsof the dermis, and there is a tendency to the formation of sheets of cells and capillaries. A moderate polynuclear infiltration is also present.
I n the second d o n k e y the inoculation g a v e rise to edematous areasr e m o t e f r o m the areas of inoculation, b u t there was v e r y little reaction
a t the inoculated sites.
Donkey 2, inoculated Nov. 30, 1926, intradermally, on the left side of the neck,and also intravenously (4 co.), with 8-day cultures of Bartonella bazilliformisgrown on blood agar plates. There was no change in the animal's temperature,
which remained slightly below or above 38°C. Rather large, fluctuating swellings
appeared within 48 hours on various parts of the body, one on the side of the back,and others on the flank and abdomen. Blood culture was negative 72 hours
after inoculation, and also after 20 days. The swellings were still present 2½months later, and one was punctured; the aspirated fluid was sterile. A pieceof tissue, removed from another "nodule" at this time for section, was histologically negative. Six weeks later a saline suspension of tissue from one of the"nodules" was inoculated into M. rhesus 30, with negative results, both as to blood
culture and local lesions. The animal's appearance was nearly normal 284 days
after inoculation. Its weight at this time was 440 lbs. Reinjection of the animal
8 months later, that is, a year and a half after the original inoculation, with live
cultures, produced the same reaction as before, and in this instance there weregeneralized small scaly eruptions all over the body, which persisted about a week.
T h e results in D o n k e y 2 were difficult to interpret, a n d two m o r edonkeys were inoculated, one with killed cultures of Bartonella b~illiformis, a n d the other with a filtrate of living cultures. T h e reactionin these two animals was m u c h the same as in D o n k e y 2. I n thea n i m a l receiving the filtrate ( D o n k e y 3) the swellings a p p e a r e d within
72 hours a f t e r inoculation (Fig. 1); in D o n k e y 4, inoculated with
heat-killed cultures, t h e y did n o t develop fully until a b o u t 10 d a y sa f t e r inoculation. I n both, however, t h e y persisted for several weeks.

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ETIOLOGY OF OROYA FEVER.

XVI

T h e s t u d y of sections m a d e f r o m t h e swollen areas yielded no exp l a n a t i o n of t h e p h e n o m e n o n w h i c h o c c u r r e d in these t w o animals.
Donkey 3, weight about 500 lbs., injected April 4, 1928, intravenously, withfiltrate (Berkefeld V) of 14-day cultures of B. bacilliformis. The temperature
was 102.4°F. late in the day of inoculation, but thereafter remained a few tenthsof a degree below or above 100°. Large raised areas developed in 72 hours, as inDonkey 2 (Fig. 1). Blood culture was negative 9 days after inoculation. Control
rhesus monkeys (injected with the same material, intravenously and intradermally)
developed no skin lesions, and blood cultures were negative. Cultures of theinoculated material also proved its sterility.
Donkey 4, 650 lbs., injected April 16, 1928, intravenously, with cultures ofBartonella bacilliformis killed by heating 30 minutes at 60°C. This material wasproved sterile by culture tests and animal inoculation. The animal's hide was
not perfectly smooth, and the natural prominences were outlined with a skinpencil before inoculation. 10 days after inoculation this animal was found to havedeveloped the same swellings as Donkeys 2 and 3, but they were less marked. Theappearance of the animal 25 days after inoculation was similar to that ofDonkey 3. A piece of tissue removed from a swelling on the left side of the animal
8 months after inoculation was found on microscopic examination to consist
solely of fat tissue.
T h e d o n k e y s used in t h e first f o u r e x p e r i m e n t s were of t h e small(440-650 lbs.) light g r a y v a r i e t y c o m m o n l y k n o w n as t h e " b u r r o . "T h e fifth d o n k e y , in w h i c h definite v e r r u g a lesions were p r o d u c e d(Fig. 3), was a larger a n i m a l (800 lbs.) a n d b r o w n in color. So far
as can be ascertained, however, t h e species is t h e same.
Donkey 5. Weight 800 lbs. Five intradermal injections were made, on Jan.
5, 1929, with a mixture of living cultures of the 5 strains of Bartondla bacilliformison hand in the laboratory,l°, n two into the skin of the left flank, two on the rightear, and one on the left ear. 10 cc. of the same mixture were injected intravenously. The material produced, on injection into the skin of the ears, swellings
about 1 cm. in diameter, which did not subside but slowly increased in size, sothat after 6 days they were 2 cm. in diameter and 12 mm. high (Fig. 3). They
were dome-shaped and slightly fluctuant. The skin over them remained intact
and was of the normal black color of the donkey's ear. The sites of inoculationon the flank secreted a clear serous liquid for a few days, without papule or nodule
formation, and then completely healed.
The animal continued to receive intravenous injections of mixed strains ofliving cultures of Bartonella bacilliformis, 20--30 cc. at each dose, twice a week
for 14 weeks, the idea being to produce, if possible, a strong local and general reaction, in the hope of obtaining an immune serum for use in other experiments.

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459

After the nodules on the ears had reached a diameter of 2 cm. they remained
stationary for a period of 3 weeks, after which they gradually became smaller
until at 72 days after inoculation they were invisible. Blood cultures were maderepeatedly throughout the period of observation and yielded uniformly negative results.
One of the nodules on the right ear was aspirated on the 22nd day after inoculation and slightly less than 1 cc. of turbid liquid obtained which proved by culture
tests to be sterile. Both nodules on the right ear were punctured three days later,
but no liquid was obtained from either. The nodule on the left ear, similar in all
respects to those on the right ear, was then removed under novocaine anesthesia.
A portion was used for inoculation of a rhesus monkey, and another portion forhistological study.
Animal passage. M. rhesus D-1 was inoculated intradermally and by scarification on Feb. 16, 1929, with a saline suspension of a part of the nodule removed
from Donkey 5. After an incubation period of 9 days typical nodules (Fig. 4)
developed at the sites of inoculation, and blood culture made the following day
was positive for Bartondla bacilliformis. Nineteen days after inoculation thenodules were mature, the area inoculated by scarification showed fully developed
lesions, and after 31 days recovery was complete. The disease ran a typicalcourse. The culture of B. bacilliformis obtained from the blood of this animal
and a subculture from it, on intmdermal inoculation into M. rhesus D-2 and D-3
produced typical reactions (Figs. 5 and 6).
Histological examination (Figs. 7 and 8). The tissue removed from the ear ofDonkey 5 consisted of the coveting epidermis, a narrow tim of normal skin encircling the nodule, and a superficial portion of the nodule itself. The latter,irregularly marked off from the rest of the tissue, was soft, gray with a faint pinktinge, and about 4 mm. thick at the center, tapering off gradually in thickness
toward the edges. The tissue was fixed in Regand's fluid, and sections werestained with hematoxylin and eosin and with Giemsa's solution.
The hematoxylin and eosin sections, examined with the naked eye, show athin normal layer of epidermis covering a layer 1 to 1.5 ram. thick of apparently
normal dermis. Beneath this is a collection of small round bodies, closely packedand tending here and there to become conglomerate. Each of these individual
nodules, varying from 1 to 3 mm. in diameter, stands out from the rest of thetissue by its deeper hematoxylin stain. Together they comprise the nodular
tissue proper.
Microscopically the minute nodules which make up the large nodule are in
general fairly distinct from one another, but in places they tend to fuse. They
are all very densely infiltrated with polynuclear leucocytes, which somewhat
obscure the underlying structure by their presence. However, numerous slit-likespaces of variable length are seen within the minute nodules, lined with large,oval, flat ceils with clear oval nuclei. These cells resemble endothelial cells.
Some of the spaces contain serum; others are empty. With higher magnification

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E T I O L O G Y OF 0 R O Y A FEVER.

XVI

there are seen larger collections of these endothelial ceils forming sheets or runningin bands, and having a tendency to form blood-containing capillary-like structures.
The overlying epidermis and the narrow zone of dermis, with its numerous hairfollicles and sweat glands, are normal. Though there is no distinct capsule, thesetissues are separated sharply from the collection of minute nodules beneath. The
surgical line of excision passes through the nodular material.
These findings show that the unit of the pathological process situated in thedeep portions of the dermis is a minute nodule, from 1 to 3 ram. in diameter. Theunits may be discrete or tend to fuse. Each is composed of a considerable proliferation of endothelial ceils, arranged in sheets, bands, and tubes. Superimposed,
there is a very marked polynudear infiltration, as was found in the section ofnodular tissue from Dog 1.
SUMMARY

ANDCONCLUSIONSIn the experiments here reported, definite verruga lesions, in which
the presence of Bartonella ba~illiformis was established b y culture orb y passage to rhesus monkeys, were produced in a dog and in a d o n k e yb y inoculation of cultures or m o n k e y passage strains. T h e reaction
induced in these animals was entirely local, however; blood cultures
were sterile. Histologically, the lesions produced were similar to
those obtained in monkeys b y inoculation of Bartonella bctcilliformis,
except for the presence of a m a r k e d polynuclear leucocytic exudate.
In another donkey a lesion histologicaUy suggestive of verruga wasproduced, while in one donkey and a horse the results of inoculation
were negative or indefinite.
T h e intravenous injection of a filtrate or of heat-killed cultures ofBartonelta bacilliformis into two donkeys was followed b y the appearance of large, soft, subcutaneous swellings, on various parts of the body,not resembling in a n y way verruga lesions.
BIBLIOGRAPHY
1. Malo, Nicolas, Anales de la Universidad de Chile, 1852, 9, 505.
2. Odriozola, E., La maladie de Carrion, Paris, 1898, p. 95.
3. Gomez, M. E., Trabajos presentados al V. Congreso Medico Latino Americano,VI. Pan Americano, Lima, Nov. 9-16, 1913, p. 118.
4. Velasquez, N. P., Ibid., p. 172.
5. Ribeyro, R., Mackehenie, D., and Arce, J., Ibid., p. 151.
6. Noguchi, H., and Batfistini, T. S., J. Exper. Med., 1926, 43, 851.
7. Noguchi, H., J. F~xper. IVied., 1926, 44, 715.
8. Noguchi, H., J. Exper. Med., 1926, 44, 697.

H. NOGUCHI~ H. R. MULLER~ E. B. TILDEN AND J. R. TYLER

461

9. Noguchi, H., J. gxper. Meal., 1927, 45, 437.
10. Noguchi, H., J. Exper. Med., 1927, 45, 175.
11. Noguchi, H., Shannon, R. C., Tilden, E. B., and Tyler, J. R., J. Exper. Med.,
1929, 49, 993.
EXPLANATION

OF P L A T E S

PLATE 21
FIG. 1. Donkey 3, 72 hours after intravenous injection with a filtrate of culturesof BartoneZla bacilliformis, showing the large swellings which arose on the side.
FIG. 2. Photograph of the same animal, taken before inoculation. For comparison with Fig. 1.
PLATE 22
FIG. 3. Appearance of nodules on right ear of Donkey 5, 39 days after inoculation. The nodule on the left ear had been partly excised 14 days previously,
and the wound was in process of healing.
FIG. 4. M. rhesus D-l, showing verruga nodules on abdominal wall 10 days
after inoculation with emulsion of nodule from ear of Donkey 5.
FIG. 5. M. rhesus D-2, 31 days after inoculation with culture of Bartonella
bacilliformis from blood of M. rhesus D-1.
FIG. 6. M. rhesus D-3, 22 days after inoculation with subculture of BartoneUa
bacilliformis from blood of M. rhesus D-1.
PLATE 23
Fro. 7. Low power magnification ( × 105) of section from nodule on ear ofDonkey 5, showing one unit of the lesion.
FIG. 8. Higher magnification ( × 600) of section from same nodule, showing
proliferation of endothelial cells, lymph and blood spaces, and polynuclear
infiltration.

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PLATE 21(Noguchi el al.: Etiology of Oroya fever. XVD

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PLATE 22(Noguchi d aL: Etiology of Oroya fever. XVI)

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PLATE 23

Photographed byLouis Schmidt(Noguchi a a2.: Etiology of Oroya fever. XVI)