野口英世著

Journal of Experimental Medicine

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T H E ETIOLOGY OF VERRUGA PERUANA.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
P~TES 2 ~o 4.
(Received for publication, September 6, 1926.)

The experimental investigation to be reported in this paper was
made possible through the cooperation of.Professor Oswaldo Hercelles, of the University of Lima, who was kind enough to obtain for me
selected material for study, and I wish at the outset to acknowledge
m y indebtedness to him and m y appreciation of his courtesy. I
wish to express m y thanks also to Professor E. Campod6nico, of the
University of Lima, through whose courtesy the material was forwarded from Lima to New York.
There are many similarities between the conditions known asverruga peruana and Oroya fever. Their geographical distribution,
which is rather curiously limited to certain narrow valleys on thewestern slopes of the Andes Mountains, between 6 ° and 13° south
latitude, is practically the same and is of the type shown by infections
conveyed by certain ectoparasites. Fever and anemia are considerably
more acute and severe in Oroya fever, but both are present in verruga.
Verruga is predominantly a chronic infection, and its conspicuous
feature is the characteristic nodular eruption on the skin which lasts
for a period of a few weeks to one of many months. Oroya fever, on
the other hand, while sometimes accompanied or followed by a similarskin eruption, is distinguished by an acute course of extreme anemia,
during which a specific intracellular parasite, Bartonella bacitliformls,
is abundantly present in the red blood corpuscles. This microorganism has rarely been seen in the blood in cases of benign verruga.
In 1910 Jadassohn and Seiffert1 showed that verruga peruana
could be transmitted to monkeys by local inoculation of suspensions
1 Jadassohn and Seiffert, G., Z. ttyg. u. Infectionskrankh., 1910, lxvi, 247.
175

176

ETIOLOGY OF VERRUGA PERUANA

of tissue from human verruga lesions, and their results have received
confirmation from Mayer, Rocha-Lima, and Werner, * and from theCommission of the Harvard School of Tropical Medicine. 3
Oroya fever has not thus far been directly transmitted to animals,
but by means of a culture recently isolated from a fatal case of thedisease4 it has been shown that young rhesus monkeys (about 2,000
gm. body weight) are susceptible to infection with BartoneUa badlliformis, though usually not to the same degree as man, and that in theinfected animals the parasite is demonstrable in its characteristic situation in the erythrocytes. In some animals which are less resistant toinfection, the extreme anemia of Oroya fever has been reproduced?
The striking fact brought out in the inoculation experiments, however, was the dual nature of the infection induced, for characteristic
signs of both Oroya fever and verruga were observed in the animals,with occasionally a marked favoring of one or the other type of disease,as the virulence of the strain became enhanced by adaptation to theexperimental animal. Systemic manifestations--moderate or marked
anemia, fever, and high titer of Bartonella bacilliformis in the blood,bone marrow, lymph nodes, and spleen--were in general more severe
in animals inoculated intravenously or intraperitoneally, and deathoccurred only in animals so inoculated, but the invasion of the bloodby the parasite was found to take place also after local inoculation.
The tendency toward the production of verrucous lesions was moremarked, as a rule, when the virus was introduced locally, and thelesions usually arose only at the sites of inoculation, as is the case whenmonkeys are inoculated directly with human verruga material. 1.~ In
one instance, however, in which passage virus was injected both locallyand intravenously, a spontaneous general eruption typical of severe
human verruga arose on various parts of the skin, remote from thesites of inoculation. The skin lesions of the animals, whether local or-~Mayer, M., Rocha-Lima, H., and Werner, H., Manch.'med. Woch., 1913, Ix,
739.
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.
4 Noguchi, H., and Battisfini, T. S., J. Exp. Med., 1926, xliii, 851.
5 Noguchi, H., J..Exp. M~., 1926, xllv, 697.

mDEYO NOOVCm

177

spontaneous in occurrence, in all instances yielded cultures of Barlonella bacilliformis, and stained sections of all such lesions revealed
the parasites in large numbers in a characteristic endothelial situation. 6
These findings, when taken in relation to the historical and epidemiological data with regard to the two conditions, e suggested verystrongly that Oroya fever and verruga peruana were both due toBartonella baciUiformis, the considerable variations in the manifestations of infection being a result of the differences in resistanceamong individuals or among different tissues of the same individual.
It was desirable, however, to approach the problem of the etiology ofverruga directly, by means of the cultural and experimental methods
used for the isolation of Bartonella bacilliformis from Oroya fever.
For the purpose of comparative study subcutaneous nodules were
removed from each of two cases of verruga in the Dos de Mayo
Hospital, Lima, placed in tubes, and covered with citrate-saline
solution (2 per cent sodium citrate in 0.9 per cent soditun chloride).
The tubes were sealed, abundant air space being allowed, and wereplaced in the ship's refrigerator during the journey to New York, aperiod of 14 days. We were fortunate in having transportation soarranged that the tissues were shipped shortly after their excisionfrom the patients and were received in the laboratory the same daythat they arrived in New York (April 7, 1926). Each specimen
was immediately examined microscopically, saline suspensions were
made, and inoculated into culture media and experimental animals,
and a portion of each tissue was fixed in Regaud's fluid for histological study.
The nodule of Case P. 5 measured about 8 X 12 X 6 mm. and was light pinkish
in color, with the exception of a small portion to which the skin was still attached.
The nodule was still firm, except for slight softening along the cut surfaces. The
citrate solution was slightly turbid, owing to liberation of tissue elements, butthere was no putrefactive odor.
Minute Gram-negative bacilli were present, occurring singly, in pairs, and inlarger groups, as well as in masses (Fig. 18). Sections stained with Giemsa's
and Gram's solutions showed masses of the organisms scattered through the tissue(Fig. 18), the general structure of which was still well preserved (Fig. 17) notwithstanding loss of cellular elements through autolysis.
Odriozola, E., La maladie de Carrion, Paris, 1896.

178

ETIOLOGY OF V'ERRUGA PERUANAThe nodule from Case J. 45 was not as well preserved. The tissue was soft andfriable, but there was no sign of bacterial putrefaction. It contained numerous
Gram-negative bacilli similar to those found in the specimen from Case P. 5 and in
addition a few small Gram-positive diplococci. In sections the bacilli were foundin the nodular tissue and the cocci near and on the skin.
Two species of minute Gram-negative microorganisms were isolated
from the tissue of Case P. 5, one indirectly, from monkeys inoculatedwith suspensions of the tissue, and one by direct cultivation. The
former is pathogenic for young Macacus rhesus monkeys and in every
respect has proved identical with the strain of Bartonella bacilliformis
previously obtained from the blood of a case of Oroya fever. The
latter, although present also in the J. 45 tissue, as shown by cultivation
experiments, is apparently a non-specific invader; it has failed toinduce characteristic local or systemic infection, and it grows within
24 hours on ordinary agar slants, while the pathogenic organism
grows slowly and only on leptospira medium or blood agar slants.
Our interest, therefore, is in the pathogenic strain obtained by inoculation of the P. 5 material.

Transmission of Verruga Peruana to Monkeys with the Excised Nodular
Tissue of Case P. 5.
Transmission of verruga peruana to monkeys, as stated earlier inthis paper, has repeatedly been accomplished by previous investigators. In the present instance, however, the tissue had been excisedfrom the patients more than 2 weeks previous to inoculation.
One young and one adult Macacus rhesus were available on April 7.
The young one was used for inoculation of the more promising material,that is, the P. 5 nodule. Another young monkey, obtained on April 10,
was also inoculated with the P. 5 material. Owing to the scarcity ofsuitable animals, the inoculations with the J. 45 material could not be
repeated.
The suspension of the P. 5 nodule induced definite systemic or local
infection in both animals inoculated; the full grown monkey receiving
inoculation of the J. 45 material showed no symptoms of infection.

M. rhesus33, inoculated Apr. 7, 1926, with a saline suspension of the nodule P. 5,
intradermally on the shaved right eyebrow and by scarification on the left eyebrow.

mlmxONOOUCm179

2 cc. of the suspension were also injected into the saphenous vein of the right leg.
After 48 hours there was a rise of temperature (Chart 1) which lasted for 2 days,
but blood taken at this time yielded no growth. On Apr. 14 the temperature rose
again and remained high for 6 days. Blood taken during the fever (Apr 16) andinoculated into leptospira medium yielded, in a 1:10 dilution, pure growth of a
microorganism indistinguishable from Bartonella bacilliformis. The sites of intradermal inoculations on the eyebrow showed some induration at this time, b u t
they never increased in size and receded within a few weeks. The lymph glandsin the inguinal and axiUary regions became markedly enlarged about Apr. 16.
Blood cultures made on two successive occasions, May 27 and June 20, 1926,
both gave pure cultures of the Barlonella-like organisms.
H,ylZ 14 16 18 ~0 ZZ ?A 26 26 60 Z 4
De,yof ~ . Z 4 6 0 10 lZ 14 16 18 Z0 Z2 X4 Z~ ~Tcmp.D ::~ :: i :: i i i i i i i i i'i i i i i i ~ i :. i i i i i I

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C'~RT 1. M. rhesus 33.
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11 1~ 1~ 1T 19 21 73 2~ Z7 ~9 i~'ofeg,~Tcmp.'F..L
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Z 4 6 8 10 1!

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CHART 2. M. rhesus 34.
M. rhesus 34, inoculated Apr. 10, 1926, with the same material and in the same
way as M. rhesus 33. A moderate rise of temperature occurred 14 days from thetime of inoculation (Chart 2), and the animal remained febrile for 4 days. Bloodcultures made on Apr. 28, 1926, yielded a pure growth of the same organism as
had been isolated from M. rhesus 33. At this time the lymph glands had becomemarkedly enlarged, but no local reactions were noticeable. Blood taken on May 12
and on May 27 was culturally positive in a 1:10 dilution. At the beginning ofJune a large subcutaneous nodule at the middle portion of the tail was noticed

180

ETIOLOGYO~VERRUGAPERUANA(Fig. 10), and on June 18 it was excised.* It showed the typical histology of verruga tissue (Fig. 11) and yielded a pure culture of the same organism as had beenobtained from the blood. Cultivation of the blood was again successful on June
21, this time with a dilution of 1:100.
T h e foregoing experiments d e m o n s t r a t e the infectivity of thev e r r u g a nodule which h a d been k e p t a t refrigerator t e m p e r a t u r e for atleast 2 weeks, and the association of infective power (as evidencedb y febrile reaction a n d the p r o d u c t i o n of a m e t a s t a t i c local v e r r u g alesion) with a definite microorganism recoverable b y culture b o t h f r o m
the blood of the animals a n d f r o m the local lesion. Blood counts
revealed no anemia in either animal, and in neither instance did theblood films contain a sufficient n u m b e r of organisms to be detected
microscopically.
Hay
:;tm~
5 7 9 1! 13 15 17 19 Zl 2~ ~ ZT Z9 31 Z 4 6 8 10 lZ 14 IO 18 Z0 ZZ
De,yofexp Z 4 6 8 10 IZ 14 16 18 z0 ~z 24 ~6 25 ~0 ~z ..M- ~ 05 ,to 4z 44 464~ 50
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CHART 3. M. rhesus 41.
T h e p a t h o g e n i c i t y of the culture o b t a i n e d f r o m the blood of M.
rhesus 33 on April 16 was tested b y inoculation of M . rhesus 41. As
the protocol shows, the culture g a v e rise not only to a local reactionb u t to a systemic infection of p r o n o u n c e d severity.
M. rhesus 41 (Fig. 1) inoculated May 4, 1926, with culture derived from theblood of M. rhesus 33 and grown for 18 days on leptospira medium. The material
was inoculated on the right eyebrow and abdomen intradermally and on the left
eyebrow and abdomen by scarification. An irregular remittent febrile reaction(Chart 3) began to be manifest 72 hours from the time of inoculation and continued
during the 2 months of observation. Induration became noticeable at the siteof intradermal inoculation on the right eyebrow after 14 days. The lesion had thegross appearance 'of a typical verruga nodule, and histological examination of the* All operations were carried out under ether anesthesia.

HIDEYO

18I

NOGUCIIIexcised tissue showed the characteristic granulomatous structure (Figs. 4, 12) andthe presence of bacilliform organisms in the endothelial cells (Fig. 13). A suspension of the nodular tissue yielded pure cultures of the organisms in a 1:10
dilution, as did also blood withdrawn on May 21. On May 27 the blood titer byculture was 1:10,000, and on June 3, 1:100,000. The nodules on the eyebrow
rapidly increased in size, becoming scarlet-red and protruding (Fig. 1). Bloodfilms made on June 3 showed numerous bacilliform bodies in the red corpuscles,
the organisms being present in the largest numbers so far observed in r h e s u s monkeys (Figs. 2, 14). Small nodules developed at the sites of intradermal inoculationon the abdomen 40 days after inoculation; at that time the nodules on the eyebrow
measured about 10 X 12 ram.
Blood counts made by Dr. J. H. Bauer showed a gradual diminution of red
corpuscles and hemoglobin and an unusually high leucocytosis:
Ery~r~ytes~rc.mm.

Hemoglobin

Leucocyt~~rc.ram.

~ef ceni

May 4 (day of inoculation) . . . . . . . . . . . . . . .
"14 (10 days after inoculation) . . . . . . . . . .
June 3 (30 . . . .
")..........
" 15 (42 """)..........
" 23 (S0 . . . . . .
)..........
" 25 (s2 . . . . . .
)..........
"
28 (55 . . . . . .
) ..........

5,088,000
4,632,000
4,760,000
4,800,000
3,392,000
3,460,000
3,552,000

8O
7O
55
4O
30
25
25

17,60023,00043,00040,00037,800

The reduction in hemoglobin, it will be noted, was relatively greater than thatin the number of red cells, a phenomenon so far not observed by us in animals
infected with the strain of B a r t o n e l l a b a c i l l i f o r m i s from Oroya fever.
Blood smears stained on June 25 revealed a peculiar appearance of the intracorpuscular parasites, which were still present in rather large numbers, but stained
reddish rather than violet, appeared thilmer, and in some instances were fragmented.
The general features of the organisms suggested that they were undergoing degeneration (Figs. 3, 15). Blood taken at this time yielded cultures in dilution of 1 :I0,
but not in 1:100. A week later no intracorpuscular parasites could be demonstrated, the animal was more active, and the nodules had decreased somewhat insize. Conditions seemed to indicate the development of a state of immunity.
No cultures could be obtained from the blood after June 25. On Aug. 11 there wasstill anemia (red blood cells 3,362,000, hemoglobin 50 per cent), but on Sept. 14
the number of red cells had increased to 4,336,000.
T h e n o d u l e e x c i s e d o n M a y 19 a n d t h e b l o o d w i t h d r a w n 2 d a y s
l a t e r were i n o c u l a t e d into two rhesus m o n k e y s . As t h e p r o t o c o l s
s h o w , b o t h m a t e r i a l s g a v e rise t o l o c a l l e s i o n s a n d also t o s y s t e m i c
infection.

182

ETIOLOGY

0FVERRUGAPERUANA

M. rhesus 42, inoculated May 19, 1926, with a saline suspension of the nodulefrom the eyebrow of M. rhesus 41, on the left eyebrow and left abdominal wall byintradermal injection and on the right eyebrow and right abdominal wall by scarification. This animal had fever for only a few days (Chart 4) during the 45 daysof observation, but cultures made with the blood on June 2 and 14, 1926, gave
pure growth of the bacilliform organisms in dilutions of 1:10 and 1:1,000 respectively.
Small reddish indurated areas along the lines of scarification on both eyebrow
and abdomen became noticeable on June 1 (Fig. 6), and 10 days later the scarified
areas appeared as linear rows of eruptions (Figs. 7, 8). Within another 10 days
the adjacent lesions had become confluent (Fig. 9) but the transverse lines werestill well separated. The intradermal inoculations in this instance, contrary to
the usual case, failed to induce any lesions.
H~y
Iune
DaZ~
20 R 24 Z5 28 ~0 1 ~ ~ T 9 11 18 15 1T 19 21
D~yofexp Z 4 6 8 10 IZ 14 15 18 20 2Z ;~4 25 26 ~0 32 M
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CHART 4.

::~i:~.ii!~lM. rhesus 42.

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Yune
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22 24 26 Z8 ~0 1 3 5 7 9 Jl 13 15 17
D~yofaxp Z 4 6 8 10 ~ 14 16 18 ~0 Z2 t4 Z6 28

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CHART 5. M. rhesus 43.
The lesions had all healed by Aug. 6, 1926.
M. rhesus 43, inoculated May 21, 1926, under ether anesthesia, with the citrate
blood withdrawn on the same day from M. rhesus 33. Intradermal inoculations
were made on both eyebrows and on both sides of the abdomen. Blood was also
applied to scarified areas on the eyebrows and abdomen, and 0.5 cc. was injected
intravenously.
The animal showed no febrile reaction at any time during the 45 days of observation (Chart 5). Blood cultures made on June 26 were negative. Small
nodules, 2 X 2 mm., appeared at each of the two sites of intradermal inoculation
within about 24 days. They showed no change during the ensuing 10 days but

m~EYoNOGUCm183

subsequently gained in size rather rapidly. They were situated subcutaneously,
wereperfectly round, and had a semitransparent, bluish,pearl-likecolor. On July 1
there were noticed about the periphery the minute red streaks which Bartonella
lesions of this type show just before vascularization. One of the nodules excisedat this time (July 1) yielded cultures in a dilution of 1 : 100,000. By Aug. 6, 1925,
the lesionshad all healed.
The microorganism obtained in culture from a monkey inoculatedwith human verruga tissue (Case P. 5) was capable, therefore, of
inducing in rhesus monkeys a local and systemic infection the manifestations of which were in all respects similar to those obtained byinoculation of the strain of BartoneIla bacilliformis from Oroya fever,i.e., a prolonged course of irregular, remittent fever, during which theintracorpuscular parasites are present in the blood, and a skin lesion
characterized by extensive proliferation of endothelial cells and by thepresence of the parasites in the cytoplasm of these cells. The infectionin the second passage animal (Monkey 41) was of the severe type;there was absolute anemia and hyperleucocytosis, and the local lesions
were very large. The organisms were present in large numbers at one
period of the disease, but only degenerated forms were found at alater stage. The blood from which the culture was obtained, however,induced only a mild, slowly developing infection, with no fever.
The local inoculation of nodular tissue from Monkey 41 into Monkey
42 (third passage) induced a severe local reaction and a febrile reactionof brief duration, during which the blood culture titer was as high as
1:1,000. As stated previously, direct microscopic detection of theparasites in the blood was unsuccessful in the case of mild infections. The cultural procedure was by far the most delicate meansof diagnosis.

Cultural and Morphological Characters of the Microorganisms from
Verruga.
The character of the lesions of M. rhesus 41 and the microscopic
findings in the blood of this animal appear to leave no doubt that theorganism injected was identical in pathogenic properties with thestrain of Bartonella badlliformis previously obtained from Oroya
fever, a conclusion which is further substantiated by cultural andmorphological study.

184

E T I O L O G Y OF VEl~RUGA P E R U A N A

The first culture was obtained on the so called leptospira medium,
which was inoculated with various dilutions of citrated blood of M.
rhesus 33 and kept at 25°C. Growth became recognizable macroscopically within a week, the uppermost layer of the medium showing
some grayish, minute particles; these gradually increased in number
during subsequent weeks (Fig. 19). Under the dark-field microscope these particles were seen to consist of numerous tightly
packed masses of irregular minute bacillary bodies. Individual organisms were occasionally seen, but the chief characteristic of theorganisms is their constant tendency to form firm aggregates difficult
to disperse (Fig. 21). Free flagella are seen in the agar mass.
On the surface of blood agar round raised colonies of variable size,
the smallest almost microscopic (Fig. 20), the largest having theappearance of fine granules, are formed within about 6 to 8 days andafter a few days reach 1 mm. or more in diameter, but seldom 2 mm.
A light grayish color is noticeable when the light falls obliquely on theculture. The colonies are firm and are peeled off the agar readily by aplatinum loop. I t is difficult to break them up in fluid to form a
uniform suspension. Under the dark-field microscope masses ofagglomerated organisms are seen to predominate (Fig. 22); a few
single o r paired individuals may be found, and these are usuallymotile. Locomotion is in one direction only, but rotatory movement is also seen.
The organisms are Gram-negative, pleomorphic, and stain unevenly
with Giemsa's solution. When stained a long time they appear
reddish and ill defined in contour; they stain much less intensely thanmost bacteria. Special staining reveals two or more flagella attached
to one end of the organism (Fig. 24), the length of the normal flagellavarying from 2 to 5 u. Abnormal detached flagella may reach alength of 20 to 30 ~.
Individuals in young cultures are more uniform in size and have adefinite contour. Short rods, 0.3 to 0.4 ~ wide and 0.45 to 1.5 ~ inlength, predominate. In older cultures the individuals are much more
irregular in size and form, and numerous granular elements measuring
less than 0.2 to 0.5 ~ are found in masses; these stain intensely andhave very indefinite outlines, the appearance simulating that ofdegenerative changes. Exceptionally long rods 2.5 to 3 u may be found.
There is no polar staining.

n~nE¥O NOOUCm

185

The optimum temperature for growth is 25-28°C.; at 37°C. growthceases within 4 to 5 days. The optimum reaction of t h e medium
lies between pH 7.8 and pH 8.
For the cultivation of this organism the addition of animal bloodor serum and hemoglobin to solid or semisolid media is essential. No
satisfactory fluid medium has so far been found. The organism is an
obligate aerobe. The cotton plugs of culture tubes m a y be impregnated with paraffin, but sealing them hermetically with sealing wax
prevents growth.
Like the strain of Bartonella bacilliformis from Oroya fever, t h eorganism is non-spore-forming and hemophilic. It is provided withTABLE I .
Antigens
Oroya

Verruga

0.5 cc.

0.5 cc.

++++
++++
++

++++
++++

m

N

c$,

0.1
0.01
0.001
0.00010m+ + + + = complete fixation.
+ + = 50 per cent hemolysis.
- -- complete hemolysis.
several unipolar flagella of the characteristic bacterial type. A distinct cell wall, such as is seen in bacteria, can be recognized inyoung, actively motile forms.
The organism does not ferment any carbohydrate so far tested.
Red corpuscles contained in the culture medium are not hemolyzed
in the course of its growth. No putrefactive process seems to occur
in the culture. Slight growth may be obtained on Loeffier's serum
medium, but no liquefaction is observed.

Serological Identification of the Verruga Strain.
The morphological, cultural, and pathogenic properties of thevermga strain resemble so much those of the strain of BartoneUa

186

ETIOLOGY OF VERRUGA PEEUANA

baciltiforrais obtained from a case of Oroya fever that the identity ofthe two organisms appeared extremely probable.
logical tests confirmed this conclusion.

Preliminary sero-

A rabbit was repeatedly inoculated intravenously with saline suspensions of cultures of Bartonella bacilliformis during a period of 4 months, 1 to 2 cc. being injected at intervals of 4 to 5 days, and the serum obtained from the animal wastested for complement fixation with its homologous strain and also with the strain
from verruga. Saline suspensions of colonies removed from the surface of blood
slant cultures after 14 days growth at 25°C., and washed twice with saline solution,
served as antigens. The suspensions were rather granular in appearance, owing
to the difficulty of breaking up the colonies. To 0.5 cc. of the antigen (this quantity did not bind complement) were added the immune serum in quantities of 0.1,
0.01, 0.001, or 0.0001 cc., and 0.04 cc. guinea pig serum as complement. Twohemolytic units of anti-sheep amboceptor were used. Controls with normal rabbit serum were made in each instance. The total volume in each case was madeup to 1.2 cc. with saline solution. The results are shown in Table I.
T h e results of the complement fixation tests show that there is an
undeniably close serological relationship between the two strains,
although the immune serum reacted more strongly with the Oroya
than with the verruga strain.

SUMMARY AND CONCLUSIONS.
A salinesuspension of a subcutaneous nodule excisedfrom a verruga
patient, and kept in the refrigeratorfor 14 days, on inoculation into
two young Macacus rhesus monkeys (Nos. 33 and 34) induced irregularfebrile reactions and enlargement of the lymph glands, and in one
instance a subcutaneous nodule arose,independently of direct inoculation,on the taft. A microorganism has been isolatedfrom the bloodof both animals, and from the experimental nodule, which in pathogenic properties and in cultural and morphological characteristicsis
indistinguishable from the strain of Bartonella bacilliformis isolated
from a case of Oroya fever.
T h e spontaneous skin lesion of M o n k e y 34 and the subcutaneous
nodules induced b y intradermal inoculation of cultures of the microorganism were histologically typical of experimental verruga lesions
in monkeys and identical with the skin lesions induced in monkeys by
Barlonella bacilliformis. T h e organism, like Bartonella bacilliformis,
is an intracellular parasite, being found in the cytoplasm of the prolif-

HXDEYO NOGUCHX

187

eratlng endothelial cells of the lesions and in the erythrocytes of theblood.
The same variations in the manifestations of disease which have been
noted in experimental infection with Bartonella bacilliformis were observed in the experimental verruga infection. In the second passage(Monkey 41) the infection induced by local inoculation of cultures
was severe both locally and constitutionally and was accompanied bymarked anemia. The organisms were found in the red cells in large
numbers. In the third passage the systemic infection was less severe,
but the local lesions were more striking.
Detection of the parasites in the blood is far more certain by thecultural method than by microscopic examination, the latter procedure being successful only in rather severe infections. The resultof blood culture is therefore the decisive method in the final diagnosis of the disease.
Preliminary serological study shows that the organism isolated in
the present instance from the skin lesion of a verruga patient and that
previously obtained from the blood of a case of Oroya fever belong to
the same serological group.
The data obtained justify the conclusion that verruga peruana iscaused by Bartonella bacilliformis. They also definitely establish
the fact that the inoculation of blood or sanguineous exudate fromlesions of verruga peruana is capable of inducing in susceptible individuals a severe febrile systemic infection, such as that to which
Carrion succumbed. The designation "Carrion's disease''6 is therefore the appropriate one for both forms of the infection.
Bartonella bacilliformis may be regarded as a bacterium, since it
has the essential features of that group of microorganisms.
E X P L A N A T I O N OF PLATES.

PLATE 2.
FxG. 1. M. rhesus 41, showing three verruga nodules on the right eyebrow 30
days after intradermal inoculatlon. The two nodules on the hmer side had arisen
at the site of removal of the initial nodule 16 days previously. Natural size.
FIG. 2. Verruga organisms in the red corpuscles of M. rhesus 41, 30 days after

inoculation. X 1,500.
FIG. 3. Verruga organisms in the red corpuscles of the same monkey 52 days

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ETIOLOGYOF VERIIUGAPEEUANA

after inoculation. They took a more reddish stain at this time and were lessdefinite in outline, probably because they were in process of degeneration. Anemia
was also most marked at this time. Subsequent attempts to find the parasite
in the blood were unsuccessful. X 1,500.
FIC. 4. Verruga organisms in the nodule of M. rhesus 41, removed 14 days after
inoculation. Giemsa's stain after fixation in Regaud's fluid. X 1,500.
FIG. 5. Verruga organisms in the nodule of a patient suffering with verruga
peruana. Courtesy of Professor R. P. Strong. Giemsa's stain, after fixation in
Zenker's fluid. X 1,500.
FIG. 6. Early lesions of experimental verruga in M. rhesus 42, 16 days after
inoculation of a scarified area on the right eyebrow with a suspension of thenodule from M. rhesus 41. Natural size.
FIG. 7. Same lesions 23 days after inoculation.
FIC. 8. Early lesions on the scarified skin of the abdomen of the same monkey
16 days after inoculation. Natural size.
FIG. 9. Same lesions 23 days after inoculation.
FIG. 10. Subcutaneous nodule which arose spontaneously on the taft of M'.
rhesus 34, 59 days after the animal had been inoculated with a suspension of anodule of the verruga patient P. 5. Natural size. For histological appearanceof the nodule see Fig. ll.
PLATE 3.
FIG. 11. Histological appearance of the subcutaneous nodule on the tail of M.
rhesus 34, 69 days after inoculation. (The gross appearance of the nodule is shownin Fig. 10.) Giemsa's stain after fixation in Regaud's fluid. × 152.
FIG. 12. Histological appearance of the nodule on the eyebrow of M. rhesus 41,
removed 14 days after inoculation. Giemsa's stain after Regaud's fixation. ×
152.
FIG. 13. Verruga organisms in the nodule of M. rhesus 41. Same section asshown in Fig. 12. X 1,000.
FIG. 14. Verruga organisms in the red blood corpuscles of M. rhesus 41. Eightdifferent fields have been placed side by side. In some of them are seen red corpuscles containing one or more organisms. Blood films made 30 days after inoculation. Giemsa'sstain. × 1,000.
FIG. 15. Blood of the same monkey 52 days after inoculation. The organisms
appear somewhat degenerated but quite numerous. Giemsa's stain. × 1,000.
FIG. 16. Bart,onella bacilliformis in the blood of an Oroya fever patient, for comparison (Patient S. A. 15, case from which B. bacilliformis was cultivated).
Giemsa's stain. X 1,000.
PLATE. 4.
FIG. 17. Verruga nodule from Case P. 5, fixed in Regaud's fluid and stained with
Giemsa's solution, 14 days after removal from patient. The characteristic verrucous structure is still recognizable, although many cells have undergone degenera-

HIDEYO NOGUCHI

189

tion. The dark masses of irregular size are aggregates of minute bacilliform organisms. X 152.
FIG. 18. The same section as that shown in Fig. 17 but under a higher magnifmation. × 1,000.
FIG. 19. Appearance of colonies of the verruga organisms in the upper portionof a tube of leptospira medium. 28 days old at 25°C. Natural size.
FIG. 20. Appearance of colonies of the verruga organisms on the surface ofhorse blood agar slant, 12 days old at 25°. Natural size.
FIo. 21. Dark-field view of the culture shown in Fig. 19. × 1,000.
FIG. 22. Dark-field view of the culture shown in Fig. 20. × 1,000.
FIG. 23. The verruga organh~ns from a blood agar slant culture, 11 days old
at 25°C. Giemsa's stain. X 1,000.
FIG. 24. Flagella of the verruga organisms. Zettnow's stain. × 2,000.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLV.

PLATE 3.

(Noguchi: Etiology of verruga peruana.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLV.

P~TE4.

(Noguchi: Etiology o[ verruga peruana.)