野口英世著

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E T I O L O G Y O F OROYA F E V E R .
XIV. THE INSECT VECTORS OF CARRION'S DISEASE.
BY HIDEYO NOGUCHI, M.D., RAYMOND C. SHANNON, EVELYN B. TILDEN,
AND JOSEPH R. TYLER.

(From The Rockefeller Instltute for Medical Research, and the Int~rna2ional Health
Dividen of the Rockefeller Foundation, New York.)
PLATES 45 TO 47,(Received for publication, December 5, 1928.)
I t is desirable to present the two parts of this investigation in a single
paper, since t h e y bear on each other so closely t h a t to publish themseparately will call for considerable repetition of statement. T h eorigin of the studies which have led to the results here presented is
to be found in the earlier papers of this series (1), and in the work of
Townsend (2), who concerned himself especially with the insect vectorof the disease embraced under the names of Oroya fever and verruga
peruana. Since the observations given in this paper were completed
after the death of Dr. Noguchi, who planned the experiments, we wish
to state briefly the circumstances surrounding the investigation.
The earlier papers of the series established Bartonella bacilliformis as thebacterial incitant of Oroya fever (Carrion's disease) and verruga peruana. The
etiology and much of the pathology of these manifestations of a single infectious
disease were made clear by the experimental studies (Nognchi) carried out between
1925 and 1928. The essential fact which remained to be determined was theprecise mode of infection in the two maladies for which the history and clinicalobservation had indicated an insect vector. Townsend's studies of the distribution of the disease and the nocturnal nature of its origin had led him to thedecision that the vector belonged to the class of pblebotomi. Indeed, he had goneso far as to designate the vector as Phlebotomus verrucarum.
The cooperation of the International Health Division of the Rockefeller
Foundation was secured in the field investigation of insects in the verruga zones in
Peru. Various insect species were collected by one of us (Shannon), identified
as far as possible on the grounds, the identifications being completed afterwards
in the United States, and sent by ship to New York, where they were tested for
infectivity on monkeys according to Dr. l~'oguchi's plans (Tilden and Tyler). The
procedure employed in this testing was as follows:
993

994

]ETIOLOGY 01~ O R O Y A

I~EVER.

X.IV

The insects were collected without the use of chemicals and sealed alive in steriletubes, which were either dry, or contained a piece of absorbent cotton moistenedwith sterile citrate solution (about half of each shipment was sent dry, the otherhalf in moist condition). Collections were made near the time of sailing of thefast boats to New York, and shipments were placed in the steamer's refrigerator
during transit.
The method of determining the presence of Bartonella bacilliformisin the insects
was to inject a saline suspension of the crushed bodies intradermaUy, sometimes
also intravenously, into monkeys (Macacus rhesus) and to make cultures of theblood at intervals of from one to four weeks later, irrespective of the occurrence of
local lesions or fever. A given lot of insects was crushed in 0.9 per cent sodium
chloride and injected intradermally into one or two sites on the shaved abdominal
skin of two monkeys at least. Because of the differences in susceptibility of
individual monkeys, duplicate tests were necessary.
The culture technique was the same as that used in earlier work on Carrion's
disease (3). The blood was withdrawn from the monkey into an equal part of 2
per cent citrate in 0.9 per cent sodium chloride, and ascending dilutions in saline(1 : 10, 1 : 100, 1 : 1,000, 1 : 10,000, and 1 : 100,000) were inoculated into the semisolid
leptospira medium (4) in amounts of 0.2 cc. One tube of the medium was inoculated with a drop of the undiluted citrated blood. The cultures were kept at
30°C. By the end of a two to three week period the positive cultures can usually
be picked out by their macroscopic appearance, but even microscopic examination
may fail to reveal a positive culture, and subculture is often desirable (5). The
growth may be so slight in the initial culture as to be easily mistaken for the haze
which develops in a tube of sterile medium after standing at 30°C.
Although Bartonella bacilliformis was detected only in the phlebotomi, it isdesirable to state that other insects collected (Shannon) in the verruga zone were
inoculated into rhesus monkeys ill the manner of the phlebotomi and the blood
cultures carried out in the same way as for the latter. In no instance was Bartonella bacilliforrMs isolated in these cultures. A list of the insects with which no
results were obtained follows:
Ticks.--Ornithodorus megnini (on burros), Argas sp.? (on burros, birds, bats),
Tick larvae (genus and species?) on lizard.
Mites.--Tarsotomus sp. (on ground), Trombidium n.sp. (on ground), Geckobia
sp. (on lizard), Geckobiella sp. (on lizard and geckos).
Lice.--Trichodectes ovis (on sheep).
Fleas.--Pulex irritans (on man), Ctenocephalus canis (on cat, dog, and man),
Rhopalopsyllus (on dog and guinea pig).

Bedbugs.--Cirnex tectularius.
Mosquitoes.--Anopheles pseudopunctipennis, Cutex quinquefasciatus (fatigans).
Buffalo gnats.--Simulium escomeli, Simullur~ sp. (on burros).
Midges.--Forcipomyla utae, Forcipemyia townsendia.
Musdd~.--Stomoxys calcitrans.
Hippoboscidae.--Mdophagus ovinus (on sheep).

H. NOGUCHI, R. C. S H A N N O N , E. B. TILDEN, A N D ~[.R. T Y L E R

995

Streblidae.--3 genera, one specieseach (on vampire bats).
Note should be made of the fact that Townsend (2) regarded the lizard as thenatural reservoir of the incitant of verruga peruana, for the reason that he detected
intracorpuscular bodies in the blood cells, which he identified with Barton's rods.
Hence the red mites(Trombidium, Tarsotoraus, Geckobia, Geckobiella), some ofwhich were obtained from geckos (Phyllodacty!usreisii), as well as the blood of two
geckos, were injected into monkeys. Culturesprepared from the blood of thesemonkeys remained sterile.
Phlebotomi of Verrugas Ca~on.
One of us (Shannon) spent from March to July, 1928, in the Rimac
verruga zone, Peru. As many varieties of insects as possible werecollected (6) from this zone and sent to The Rockefeller Institute in
New York to be tested upon monkeys. It is desirable to state thatTownsend was the first to implicate phlebotomus with the transmissionof verruga peruana. His studies, conducted between 1912 and 1914,
led him to decide, on ecological and experimental grounds, that a
species of gnat, later called Phlebotomus verrucarum Townsend, wasthe vector of the disease.
Our studies revealed three species of Pktebotomus in the verruga
zone. Two of the species had a wide and the third a limited distribution only in the zone. I t is significant t h a t of the three, only the two
which occurred in considerable numbers were found on inoculation
to yield Bartonella baciltiformis.
In this paper brief descriptions only will be given of the three species,
based upon the characters of the males. All three species belong to thesubgenus Brumptomyia (Fran~a and Parrot), which may be describedas follows:
Basal segment of the upper appendage of the male terminalia with a weUdefined
sub-basal tuft of hairs on the inner surface; the distal appendage either with fourwell developed spines, the fifth one weak, or with five strong spines; median appendage simple, without spines, lower appendage unarmed. The abdominal hairs
are suberect to erect; length of the upper branch of upper forked vein longer thanthe petiole preceding the fork.
Ph. noguchii and Ph. peruensis are describedhere for the first time (Shannon).

Key to the Males.
1. Distal segment of upper appendage with four well developed spines, thefifth (an apical one) being very slender and hair-like; petiole of upper forked

996

ETIOLOGY OF OROYA FEVER.

XIV

cell slightly longer than that section of the first longitudinal vein which
overlaps the upper branch of the second vein . . . . . Ph. verrucarum Townsend.
2. Distal segment with five well developed spines, both apical ones being equallystrong.
(a) Distal segment with two submedian spines, a third located slightly distalof the middle of the segment, the fourth and fifth forming an apicalpair; petiole of the upper forked cell much longer than that section of
the first vein which overlaps the upper branch of the second vein. (Typelocality, Verrugas Cation, Department of Lima, Peru)
Ph. noguchii Shannon.
(b) Distal segment with two submedian spines, a subapical one and an apicalpair; that section of the first vein overlapping the upper branch of thesecond is distinctly longer than the length of the petiole of the upper
forked cell. (Type locality, Matucana, Department of Lima, Peru)
Pk. peruensis Shannon.
The females of Phlebotomus peruensis can be separated from those of verrucarumand noguchii by differences in the arrangement of the wing veins, but definite
characters have not yet been found whereby the females of verrucarum and noguchii
may be positively identified. Approximate identification of the females whichwere sent to New York for bacteriological study was made on the basis of (1)average differences in size, verrucarum being in general smaller than noguchii,and (2) habitat. All males found in houses proved to be verrucarum, hence all
females found in-doors w~re assumed to belong to this species. All the noguchii
males were found out-of-doors, in excavations and natural cavities, where they
were three times as numerous as verrucarura males.
Before leaving this description, it m a y be well to bring together
certain accepted facts with reference to the epidemiology of Carrion's
disease and certain known habits of the phlebotomi. I t is admitted
t h a t the disease is contracted only in certain limited areas in Peru, andt h a t infection, with possibly rare exceptions, takes place only at night.
This infection m a y be acquired indoors or in localities remote fromh u m a n habitations and at any time of the year.
W i t h these facts in mind, it would seem to follow t h a t the insect
vector must be (a) common blood sucker of man; (b) restricted to theverruga zone; (c) nocturnal in habit; (d) capable of breeding in varied
localities, so t h a t adults, which have restricted flight, m a y be everywhere present, and (e) active throughout the year.
All these conditions are fulfilled b y phlebotomi and not, as far as
determined, b y other insects of the verruga zones.
Finally, it m a y be recorded t h a t we (Shannon and assistant) safely

H. NOGUCHI~ 1%. C. S H A N N O N , E. B. TILDEN, A N D ~. 1%. T Y L E R

997

spent from 2 to 3 nights a week for 4 m o n t h s in the verruga zone
after taking adequate precaution to protect ourselves from bites ofphlebotomi.
Experiments with Phlebotomi.

Eighteen special lots of phlebotomi, prepared and shipped as
described, were inoculated into monkeys (Tilden and Tyler). T h ematerial in each instance was introduced intradermally at several sites
on the shaved skin of the abdomen, and was also applied to a scarified
area of the abdominal skin. Occasionally an intravenous injection
was also made.
Lots 1, 2, 9, and 14 were pooled, ground in a mortar with 0.9 per cont salinosolution, and injected, Apr. 25, 1928, into two monkeys (Macacus rhesus I-3 andI-4).
Lot 1, collected Mar. 9, 1928, out-of-doors in Matucana. 3 females and 4 malesof Ph. noguchii, Ph. verrucarum, and Ph. peruensis.
Lot 2, collected Mar. 9, 1928, out-of-doors in Matucana. 2 females, 18 males,
Phlebotomus, chiefly noguchii.
Lot 9, Ph. verrucarum collected Mar. 20, 1928, in house in Verrugas Cation.
40 females.
Lot 14, Pit. verrucarum, collected Mar. 26, 1928, in Verrugas Cation. 24
females.
Lot 20. Pk. noguchii, with possibly a few verrucarum intermixed, collected Apr.
9, 1928, consisted of about 20 females. A saline suspension of the crushed insects
was injected May 31, 1928, into two monkeys (M. rhesus I-7 and I-8). I-8 also
received 1 cc. of the suspension intravenously.
Lots 27 and 28. Ph. noguchii, with possibly a few verrucarum intermixed, collected May 1 and May 8, 1928. 10 to 12 females. A saline suspension of thecrushed insects was injected June 13, 1928, into two monkeys, M'. rhesus 1-17
and I-5.
Lots 29, 30, and 38. Ph. verrucarura, collected May 1 and May 8, 1928, 15 to
20 females. Saline suspension injected June 13, 1928, into M. rhesus 1-16 andM. rhesus I-6.
Lots 40, 41, and 44. Pk. verrucarum, collected June 9, 11, and 18, 1928, bothin houses and out-of-doors, in Verrugas Cation. These lots comprised about 100
females. Saline suspension injected July 14, 1928, into M. rhesus 1-26 and 1-27.
All the specimens which came in moist condition were covered with green mold.
Lots 39 and 45. Ph. noguchii, collected June 6 and June 19, 1928, out-of-doors
in Verrugas Cation. The number of insects was small (about 25 females), and thespecimens which came in moist condition were covered with green mold. Saline
suspension inoculated July 14, 1928, into M. rhesus 1-28 and 1-29.

998

ETIOLOGY

OF OROYAFEVER.

XIV

Lots 42 and 46. Ph. peruensis, collected June 12 and 19, 1928, out-of-doors in
Matucana. The specimens which came in moist condition were covered withgreen mold. 8 females. Saline suspension inoculated July 14, 1928, into M.
rhesus 1-30 and 1-31.
Lot 43. Ph. noguchii, with possibly a few verrucarum intermixed, collected
June 12, 1928, out-of-doors in Matucana. Saline suspension inoculated Aug. 13,
1928, into two monkeys, 1-33 and 1-34.
Lot 51. Ph. verrucarum, collected during the last week in July, 1928. 6
females. Saline suspension inoculated Aug. 13, 1928, into M. rhesus 1-37.
Lot 54. Pk. noguchii, with possibly a few verrucarum intermixed, 8 to 10
females, collected during the last week in July out-of,doors in Verrugas Cation.
Saline suspension inoculated Aug. 14, 1928, into M. rhesus 1-38 and 1-39.
T h e first material inoculated, which contained all three species of
Phlebotomus (Lots 1, 2, 9, and 14) yielded positive results.

Strain 1, from Lots 1, 2, 9, and 14.
M. rhesus I-3 and I-4, inoculated intradermally Apr. 25, 1928. No local lesions
developed at the sites of inoculation. M. rhesus I-3 had a temperature of 104°F.
on May 14, 19 days after inoculation, and blood was withdrawn on that day.
Bartonella bacilliforeais was obtained in culture from 1 : 10, 1 : 100, and 1 : 1,000
dilutions of the blood. The temperature reached 104°F. again several times, butblood culture was negative on May 29 and June 30. Blood was taken from M.
rhesus I-4 at the same time as from I-3, but cultures remained negative.
Inoculation of Cultures from M. rhesus I-3.--M. rhesus 1-14 and M. rhesus 1-15
(Fig. 1) were inoculated June 5, 1928, with the 20 day culture of Bartonella bacilliformis obtained from the blood of M. rhesus I-3 and a 5-day subculture. The
culture, which was, as usual, diluted with an equal part of saline for inoculation,
was also applied to a scarified area on the abdominal skin. Tiny nodules wereobserved in M'. rhesus 15 at the sites of intradermal inoculation on June 11 (16days after inoculation), and 5 days later they were well developed, and one was
excised1 for examination and transfer. Bartonella bacilliformis was obtained
from a 1 : 1,000 dilution of blood withdrawn June 16, and from a 1 : 100,000 dilutionof the nodule suspension. By June 22 the scarified area showed small nodules.
The photograph was taken 3 days later (Fig. 1). By June 28 the lesions had
disappeared. M. rhesus 1-14 had almost continuous high fever (104° to 106°F.)
from June 8 to 28 and again from July 16 to 23, but no local lesions developed,
and blood culture was negative 13, 31, and 55 days after inoculation.
The strain of Bartonella bacilliformis obtained from these first lots ofphlebotomi was carried through two animal passages b y direct transfer
and has since been maintained b y alternate generations in culture and1All operations were carried out under ether anesthesia.

H. NOGUCHI~ R. C. SHANNON~ E. B. TILD:EN~AND J. R. TYLER

999

monkey. T h e usual course of verruga of moderate severity (7) has
been observed in the animals (Fig. 8), with the exception of one
m o n k e y of the first passage (M. rhesus I-1), which had an unusually
severe cutaneous reaction (Figs. 6, 7), not unlike t h a t which had been
induced in one of the monkeys (M. rhesus 18) of an early experiment(Noguchi (8)). This animal was acutely ill over a period of two weeks
b u t recovered.
Histological study of the nodular tissue from 1-15 and I-l, made b y
Dr. H e n r y R. Muller, shows the characteristic proliferation of endothelial cells and the formation of new capillaries. Bartonella bacilliformis was detected in some instances within the endothelial cells.
T h e second lot of phlebotomi tested, which probably consisted
chiefly of Ph. noguchii b u t m a y have contained a few verrucarum,
also yielded a strain of Bartonella bacilliformis.

Strain 2 from Lot 20.
M. rhesus I-7 and I-8 were inoculated May 31, 1928, intradermally, and I-8
received 1 cc. of the suspension in the left saphenous vein. The animals were bledon June 11 and again on June 30. The blood of I-8 yielded cultures of Bartonella
bacilliformis in 1:100,000 dilution on June 11 and in 1 : 100 dilution on June 30; nocultures were obtained from the blood of I-7. Neither animal developed lesions
at the sites of inoculation, but I-8 had almost continuous fever (104° to 105.2°F.)
from June 4 to June 28.
Inoculation of Culture from M. rhesus I-8.--M~. rhesus 1-22 and 1-23 were inoculated on June 22, 1928, intradermally, with a 10-day-old culture from the bloodof M. rhesus 1-8. The culture was also applied to a scarified area on the abdomen.
The nodules showed at the sites of intradermal inoculation after about 10 days,
and blood withdrawn after 2 weeks yielded cultures of Bartondla bacilliformisin
1 : 10,000 dilution. The nodule excisedI on July 6 for examination and transfer
yielded cultures in a 1:100 dilution. The lesions were considerably larger andmore extensive in 1-23 (Fig. 3), and the edema of the abdominal wall developed
early and became marked. Regression of the lesions began about four weeks
after inoculation and within four weeks recovery was practically complete. The
course of disease was almost afebrile in both animals.
F u r t h e r inoculations, first with nodular tissue from 1-22 and 1-23,
and later with cultures from the blood of passage animals, showed t h a t
this strain of Bartonella bacilliformis, like Strain 1, was moderately
virulent, inducing pronounced local lesions and moderate anemia.

I000

ETIOLOGY

OF O R O Y A

FEVER.

XIV

One animal of the series died in 33 days, after an afebrile course of
disease, during which moderately severe anemia h a d been observed.
Lots 27 and 28 (chiefly Ph. noguchii) and Lots 29, 30, and 38 (Ph.
verrucarum) yielded negative results, as did also Lots 40, 41, 44, a n d
51 (Ph. verrucar~m), Lots 42 and 46 (Ph. peruensis), and L o t 43 (Ph.

noguchii).
F r o m Lots 39 and 45, which p r o b a b l y consisted of Ph. noguchii

alone, a third strain of Bartonelta bacilliformis was obtained.
Strain 3, from Lots 39 and 45.
M. rhesus1-28 and 1-29were inoculated intradermaly on July 14,1928. Monkey
1-28 showed no fever at any time, and b ood cultures were negative 1, 2, and 3
weeks after inoculation. Monkey 1-29 had fever (104.2° to 105.2°F.) 3 days after
inoculation, which continued for a week with one day of remission. The Moodyielded cultures of Bartonella bacilliformis in a dilution of 1 : 100 on four occasions,7, 13, 23, and 31 days after inoculation, but there was no reaction at the sites of
injection.
Inoculation of Culturesfrom 1-29.--M. rhesus 1-44 was inoculated intradermallyand by scarification on Aug. 16, 1928, with a culture 14 days old from the blood of
M. rhesus 1-29. 1 cc. of the culture was also injected into the saphenous vein.
Nodules developed in 2 weeks (Figs. 5 and 9), and the scarified area presented thecharacteristic miliary eruption. The blood was positive in a dilution of 1 : 10,000
at this time. From Sept. 7 to 19 there was marked fever (104° to 105.2°F.).
M. rhesus 1-45 was inoculated at the same time and in the same manner as
1-44. The intradermal nodules attained a diameter of only 0.5 cm., and the blood
was positive in a dilution of 1 : 100. Fever existed (104.2° to 105.4°F.) from Sept.
10 to 13, was followed by two days of subnormal temperature, and death of animal
on Sept. 16. Autopsy (Dr. Muller) revealed nothing abnormal except in the
spleen, which contained numerous pale areas 2 to 3 ram. in diameter. Film
preparations were negative for tubercle bacilli, and microscopic examination
disclosed infarcts such as are found in the sp'een in human (9) cases of Oroya
fever, and in cases of the experimental disease (10).
L a t e r passage of Strain 3 produced local lesions of very large size
(2 to 3 cm. in diameter), b u t no unusual systemic effects.
L o t 54, which consisted chiefly, perhaps wholly, of Ph. noguchii,also yielded Bartonella bacilliformis.

Strain 4, from Lot 54.
M. rhesus 1-38 was injected intradermally on Aug. 14, 1928, and intravenously(1 cc. of the saline suspension into the left saphenous vein). From Aug. 22 toH. N O G U C H I , ]i. C. S H A N N O N , E. B. TILDEN, A N D J. R. T Y L E R

I001

Aug. 29 the temperature was 104°F., but blood culture made Aug. 28 was negative.
It was also negative on Sept. 10, but blood taken on Sept. 25, when the temperature was 104.2°F. yielded Bartonella bacilliforrais in 1 : 10 and 1 : 100 dilutions
after 13 days incubation. The intradermal mixtures produced no lesions. M.
rhesus 1-39, inoculated at the same time as 1-38, and with the same material,
showed a rise of temperature (104.2° to 104.8°F.) on three occasions, but blood
cultures made on Aug. 28, Sept. 10, and Sept. 25 were negative.
Inoculation of Culturesfrom 1-38.~M. rhesus 1-58 was inoculated on Oct. 10,
1928, with 15-day culture from the blood of M. rhesus 1-38. Small nodules
appeared at the sites of intradermal injection after 7 days and were well advanced
after 16 days (Fig. 5). The abdominal wall became oedematous and the area of
scarification showed miliary nodules in addition to which three or four small
eruptions arose outside the inoculated areas. Blood culture was positive in
dilutions up to 1 : 10,000, 12 days after inoculation. The animal died on the 18thday, when the local lesions were still actively progressing. Histological examination of tissues by Dr. Muller revealed the characteristic zonal necrosis around thecentral vein in the liver, with extensive invasion by polymorphonuclear leucocytes.
The spleen showed no lesions. The various skin nodules were histologicallycharacteristic of verruga in the monkey.
F u r t h e r inoculations with cultures of Strain 4 fielded similar results.
In one animal (M. rhesus S - 7 ) t h e local lesions reached large size.
T h e results of the inoculations are summarized in Tables I to V.

Exposure of Monkeys to Bites of Phlebotomi.
Six rhesus monkeys were exposed (Shannon) for several weeks t onatural infection, three in an excavation in Verrugas Cation, where
Ph. noguchii was fairly common, and three in a house where Ph.
verrucarum was abundant. These animals were brought to T h e
Rockefeller Institute on Aug. 13. Blood withdrawn on three occasions
failed to yield cultures of Bartonella bacilliformis, and only one of theanimals failed to respond to subsequent inoculation of virulent cultures
or passage virus. T h e result therefore was regarded as negative.

Immunity.
Seven of the monkeys which had developed verrucous lesions andblood infection with Bartonella bacilliformis following inoculation with
the Phlebotomus strains and in which the lesions had regressed, were
subsequently tested for i m m u n i t y b y reinoculation. Similar imm u n i t y tests were made on three monkeys which had received crushed

1002

ETIOLOGY O F OROYA F E V E R .

XIVTABLE I.

Inoculations of Crushed Ptdebotomi.
M. rhesus
No.

Da~
1928

I-3

Apr. 25

Lot No.

Method of inoculation

Local
lesions+

Multiple intradermal
Scarification

1, 2, 9, 14

Bloodculturenoguchii
peruensis
I4
I-7

Same

May 31

Same
2O

Same

noguchii (few verrucarum?)
L8
1-16

Same

Same

June 1329, 30, 38

Same, also intravenous
Multiple intradermal
Scarification
Same
Same

7)err~RTaTu~

I-6
1-17

Same

Same

Same
27, 28m

Multiple intradermal
Scarification--+

noguchii (few verrucarum?)
I-5
1-26
1-27
1-28

July 14Same
40, 41, 44

Same~rTucar~ff$
Same

Same

Same

39, 45

Same

Same

Same
w

Same

noguckii
1-29
1-30

Same

1-31
1-33

Same

Same

Same
42, 45

+

Same

Same

peruensis
Aug. 13Same
43

noguchli (fewrucarum?)
1-34
1-37

Same

Same

Same

Same

M

m

~er-

Same
51

Same

Same

m

v~r/'ucaru~

1-38

Same

54

Same, also intravenous

noguchii (few verrucarum?)
1-39

Same

Same

Same

+

H. NOGUCHI~ R. C. SHAN~NON~E. B. TILDEN~ AND J. R. TYLER

1003

TABLE II.

Strain 1, from Lots 1, 2, 9, 14 (Pk. ~errucarum, Pk. noguckii, Pk. peruensis).
I

M. rkesus
No.

Date
1928

Material inoculated

1-14

June 5

Culture from I-3

1-15

Same

Same

Mode of inoculation L o c a l lesions
Intradermal
Scarification
Same

Blood
culture

+++

+

++++
++++
++++

+
+
+

++++
+++

+
+

First passage
1-18
1-19
I-1

June 16
Same
Same

Nodule susp. 1-15
Same
Same

Same
Same
Same

Second passage
1-12
1-13

!

July 6
Same

Nodule susp. I-1
Same[ Same]S a m e

Third passage (via culture)
1-40

Aug. 16

14-day culture 1-12

1-41

Same

Same

Same, also intravenous
Same

++++[
++++

+

TABLE III.

Strain 2 from Lot 20 (Pk. noguckii--few verrucarum?).
M. rhesus
No.

Date
1928

1-22

June 22

1-23

Same

Material inoculated

10-day culture from
I-8
Same

Mode oi inoculation
Intradermal
Scarification
Same

Local lesions

Blood
culture

++++

+

++++

+

++++
+++

+
+

First passage
1-24
1-25

July 6
Same

Nodule susp. 1-23
Same

[ SameI SameSecond passage (via culture)
1-42

Aug. 16

1-43

Same

14-day culture from
1-24
Same

Same, also intravenous
Same-{-+-}--~+ + +-Jr-

1004

ETIOLOGY

OF OROYAFEVER.

XI'V

TABLE IV.

Strain 3 from Lots 39 and 45 (Pk. noguckii).
M. rt~sus

No.

Date
1928

iMaterlal inoculated

Mode of inoculation~c~ ~ons

Blood
cultureI--44

Aug. 16

14-day culture from
1-29

++++

+

1-45

Same

Same

Intradermal
Scarification
Intravenous
Same

++++

-~-

+++

+

First passage
1-55

Sept. 13

Nodule susp. 1-44

1-56
1-34

Same
Oct. 22Same
20-day culture from
1-45

Intradermal
Scarification
Same
Same

+

Second passage via culture)
S-6

I Dec. J5

25-day culture from
1-34

Same

++++

I

+

TABLE V.

Strain 4 from Lot 54 (Pk. noguckii--/ew verrucarum?).
rhesus ][•M.__N°'

Date
1928

I~858 [ Oct. 10

tMaterial inoculated

Mode of inoculation

Local lesions

Blood
culture15-day culture from
1-38

Intradermal
Scarification

+++

+

++++

+

++++

+

First passage via culture)
1-17

Nov. 514-day culture from
1-58

Same

Second passage via culture)S-7

Dec. 15

18-day culture from
1-17

Same

H. NOGUCHI, R. C. SHANNON~ E. B. TILDEN~ AND ~'. R. TYLER

1005

TABLE VI.

Immunlty Tests.
First inoculation
M. rhesusNo.

Immunitytest

--

-

A

1-18

June 16 Nodulesusp. -l-{--l-}-+nu +1-15 (Str.

Sept. 13

Nodulesusp.
1-41 (Str.

1-13
I-3

July 6
Apr. 25

Same
Same

--

--

Same
Same

++++

--'
--

Same

Same

++++

+

+

Sept. 13

+

1>

1}

Same
++++
Phlebotomi-Lots 1, 2,
9, 14

+
+

1-53
Control
I-8

May 31

Phlebotomi
Lot 20

1-25

July 6

Nodulesusp. - { - + + +1-23 (Str.

+

Same

l~Iodulesusl~ - t - + + +1-43 (Str.
2)
Same
-

1-23

June 22

Same

Same

--

--

1-11

July 6

Same

Same

--

-

286
1-54
Control
I-1

June 1

Blood culture + + + - t Jr
I-8 (Str. 2)
Nodulesusp. + + + ++P. 5*
Culture P. 5* -I-+-l++ +

Same
Same

Same
Same

+++

+

+

Sept. 26

Culture
P. 5*

-

--

+
+

Same
Same

Same
Same

+++

+

Same

Same

+ Jr +

-[-

--

-

2)

June 16 Nodulesusp. - t - + + +1-15 (Str.

1)
1-19
1-29

June 16 Same++++
July 14 Phlebotoml
Lots 39, 45

1-57
Control* Noguchi, H., J. Exp. Med., 1927, xlv, 175.

phlebotomi without developing skin lesions which reacted as do previously untreated animals. Table VI summarizes the results.

1006

ETIOLOGY

OF OROYAFEVER.

XIV

Morphology.
No morphological or cultural differences could be detected between
the Phlebotomus strains and the human strains of Bartonella bacilliformis. Cultures seven days old of the four strains, grown on horse
blood agar slants, were stained to bring out the unipolar flagella (oneto four) which are characteristic of Bartonella bacilliformis (Figs. 11,
13, 15, 17), the films being made on the same slide, in order that thestained preparations might be comparable. Cultures of the same age
but grown on leptospira medium were used for similar comparative
preparations which were stained by Gram's method, with fuchsin as
the counterstain (Figs. 10, 12, 14, 16).
SUMMARY AND CONCLUSIONS.
With a view to determining the mode of infection in Carrion's

disease, a study of the blood-sucking insects found ir~ the districts ofPeru where the disease prevails has been carried out, through the cooperation of The Rockefeller Institute and the Rockefeller Foundation. The material studied included ticks, mites, midges, lice, fleas,
bedbug% mosquitoes, buffalo gnats, horse-flies, "sheep ticks," 3
species of Streblidae, and 3 species of Phlebotomus, including Phlebotomus verrucarum Townsend and two new species which have been
named Phlebotomus noguchii and Phlebotomus peruensis. The insects
were collected without the use of chemicals, were prepared for transportation in such a manner as to prevent drying, and were shipped
under conditions of refrigeration to New York, where they were
inoculated into monkeys. The plan followed was to inject saline
suspensions of the crushed insects intmdermally into rhesus monkeys
and to make cultures of the blood of the animals at intervals of 1 to 6
weeks after inoculation.
The only class of insects in which the presence of Bartonella bacilliformis could be detected were phlebotomi. No cutaneous lesions were
induced in monkeys injected with the crushed insects, but in the caseof four different lots of phlebotomi the blood of the animals so injected
yielded cultures of Bartonella bacilliformis which produced typicalverrucous lesions on inoculation into other monkeys.
The morphology and cultural characteristics of the Bartonella strainsobtained from phlebotomi proved identical with those of strains

H. NOGUCHI, R. C. SHANNON, E. B. TILDEN, AND ~. R. TYLER

1007

isolated from human blood and skin lesions. Monkeys which had
recovered from infection with the phlebotomus strains resisted inoculation with a human strain of Bartonella badlliformis, and, conversely, monkeys which had passed through an infection induced b y
the human strain resisted inoculation with the strains obtained fromphiebotomi.
The experimental observations described in this paper lead us toconclude that certain phlebotomi act as insect vectors of Oroya fever
and verruga peruana. The phlebotomi which have been shown quite
certainly to carry the Bartonella bacilliformis are those of the species
Phlebotomus noguchii. Phlebotomus verrucarum is also probably avector, while Phlebolomus peruensis remains doubtful in this respect.
BIBLIOGRAPHY.
1. Noguchi, H., and Battistini, T. S., J. Exp. Med., 1926, xliii, 851. Noguchi,
H., Ibid., 1926, xliv, 533, 697, 715, 729; 1927, xlv, 175, 437, 455, 781; 1928,
xlvii, 165, 219, 235; xlviii, 619. Noguchi, H., Shannon, R. C., Tilden,
E. B., and Tyler, J'. R., Science, 1928, lxviii, 493.
2. Townsend, C. H. T., Peru Today, 1913, v, 840; J. Am. ivied. Assn., 1913, lxi,
1717; West Coast Leader, Mar. 8, 1927.
3. Noguchi, H., J. Exp. ~ed., 1926, xliv, 533,697,729; 1928, xlvii, 219.
4. Noguchi, H., Muller, H. R., Torres, O., Silva, F., Martins, H., Ribeiro dos
Santos, A., Vianna, G., and BiAo, M., Monograph of The Rockefeller Institute for Medical Research, No. 20, 1924, p. 10.
5. Noguchi, H., J. Exp. Med., 1926, xliv, 537.
6. Shannon, R. C., Am. J. Bryg., in press.
7. Noguchi, H., J. Exp. Med., 1927, xlv, 455.
8. Noguchi, H., J. Exp. Med., 1926, xliv, 697.
9. Strong, R. P., Tyzzer, E. E., SeUards, A. W., Brues, C. T., and Gastiaburfi,
J. C., Report of first expedition to South America, 1913, Harvard School of
Tropical Medicine, Cambridge, 1915.
10. Noguchi, H., J. Exp. Med., 1927, xlv, 437.
EXPLANATION OF PLATES.
PLATE 45.

FIG. 1. Cutaneous lesions induced in M. rhesus 1-15 by Phlebotomus Strain 1.
Photograph taken 20 days after inoculation. One intradermal nodule had been
excised 2 days previously.
FIG. 2. The appearance of the lesions in M. rhesus I-1, a Strain 1 first passage
animal, 18 days after inoculation. The sacrified area already shows characteristic
minute nodules, All the lesions reached considerable size (Figs. 6 and 7).

1008

ETIOLOGY Or OROYA FEVER.

XlV

Fro. 3. Strain 2. Early culture lesions (two weeks after inoculation) in M.
rhesus 1-23.
FIG. 4. M. rhesus 1-44, 21 days after inoculation with cultures of Strain 3 from
Lots 39 and 45 of Ph. noguchii.
FIG. 5. M. rhesus 1-58, 16 days after inoculation with Strain 4 phlebotomus

cultures. The eruption was more general and the edema extensive in this animal.
Death occurred 3 days after the photograph was made.
PLATE 46.
FIGS. 6 Am) 7. Late lesions in M. rhesus 1-1 (Strain 1) as they appeared 31 days
after inoculation. The most pronounced lesion occurred at the scarification site
(center).
FIG. 8. M. rhesus 1-19, 29 days after inoculation in the same way and at the sametime as M. rhesus 1.
FIG. 9. M. rhesus 1-44, 21 days after inoculation with cultures of Phlebotomus
noguddi Strain 3.
PLATs 47.
Magnification × 1,000.
FIG. 10. Plflebotomus Strain 1, from Lots 1, 2, 9, 14. Gram's stain, counterstained with saturated alcoholic solution of fuchsin.
FIG. 11. Same, stained for flagella, by a combination of Zettnow's mordant andFontana's ammoniac silver solution.
FIG. 12. Phlebotomus Strain 2, from Lot 20. Gram's Stain, counterstained with
fuchsin.
FIG. 13. Same, Zettnow-Fontana flagella stain.
FIG. 14. Phlebotomus Strain 3, from Lots 39 and 45. Gram's stain, counterstained with fuchsin.
FIG. 15. Same, Zettnow-Fontana flagella stain.
FIG. 16. Phlebotomus Strain 4, from Lot 54. Gram's stain, counterstained with
fuchsin.
FIG. 17. Same,Zettnow-Fontana flagella stain.

THE JOURNAL OF EXPERIMENTAL MEDICINEVOL. XLIX.

PLATE 45.

(Noguchi a a/.: Etiology of Oroy~ fever. XIV.)T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XLIX.

PLATE 4.6.

(Noguchi el al.: Etiology of Oroya fever.

XIV.)THE JOURNAL OF EXPERIMENTAL MEDICINE VOLo X L I X .

PLATE 47.

(Noguchl et a/.: Etiology of Oroya fever. XIV.)