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Journal of Experimental Medicine

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E T I O L O G Y OF OROYA F E V E R .
IX. BACTERIU~ PERU'VIANUM,N. SP., A SECONDARYINVADER OF THE
LESIONS OF VERRUGA PERUANA.
BY HIDE¥O NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
PLATE 6.

(Received for publication, November 3, 1927.)
In the course of an experimental investigation of Carrion's disease,at the stage when bacteriological and experimental study of the characteristic lesions of verruga peruana was desirable, I was so fortunate
as to obtain, through the kindness of Professor Oswaldo Hercelles, ofLima, two subcutaneous nodules, excised under aseptic conditions,from two verruga patients (Cases P 5 and J 45), and forwarded inseparate sealed tubes in the ship's refrigerators from L i m a to New
York. A strain of Bartonella bacilliformis was isolated from one of
the nodules (Case P 5), as has been reported elsewhere, 1 but anothermicroorganism was also present in the tissue, and was isolated in purecultures in both instances. This organism is particularly interesting
because of its striking morphological similarity to Bartonella bacilliformis and its marked difference from this parasite in cultural andpathogenic properties.

Cultural Properties.
The microorganism in question grows rapidly on ordinary bacteriological culture
media at 370C. as well as at 25°C., the latter temperature being the optimum one.
Round, discrete, elevated, grayish, translucent colonies measuring 1 to 2 nun. indiameter make their appearance on a plain or blood agar plate within 24 hours.
They reach a size of 2 to 4 mm. within several days but seldom become muchlarger. The colonies are spherical, and the margin smooth; in consistency they
are somewhat firmer than colonies of B. coli or B. proteus, and they show no1Noguchi, H., J. Exp. Med., 1927, xlv, 175.
165

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ETIOLOGY OF OROYA FEVER. IX

tendency to spread over the surface of the medium. In 7 to 10 days at room
temperature a trace of brownish tint appears in colonies on the surface of agar.
On blood agar plates hemolysis takes place and gradually extends to a considerable
distance around the colonies. A characteristic pungent odor is noticeable when
the plate is uncovered. Broth is diffusely clouded by the growth of the organism,and within a few days a delicate grayish peUiele is formed around the wall of theculture tube at the top of the fluid and a fluffy sediment at the bottom. As theculture grows older the broth becomes markedly mucilaginous. No putrefactive
or other odor is produced either in broth or on plain agar. LSff[er's serum undergoes slow but complete liquefaction within about 2 weeks at 25°C. No carbohydrates are fermented by the organism, and no hydrogen sulfide is produced on anymedium. Cultures remain transplantable for a period of at least 3 months if
kept constantly at 25°C. No growth takes place under strictly anaerobicconditions.

Morphology.
Individuals in young colonies on an agar surface are actively motile andmeasure about 0.3 to OAt* in width and 0.6 to 1.2/~ in length. In older cultures
longer forms, 2 to 4~, are present. When grown on a blood agar slant or on
leptospira medium the organisms are somewhat thinner (0.2 to 0.3/, in width) andlonger than the young forms on an agar surface and give the impression of delicate
slender rods (Fig. 3). They are less actively motile and may be clumped into
masses of many individuals (Fig. 1) which resemble to some extent the characteristic masses of Bartonella bacilliforva{s. Forms grown on broth are of medium size
and remain actively motile for many days. In cultures several days old the motileorganisms are intermingled with small oval or coccoid forms which may be inpairs or short chains. Except for occasional very long individuals, which may bebent and thickened at one end, the organisms are as a rule straight and havepointed ends. No bifid or branching individuals have so far been noted.
The organism is Gram-negative and stains fairly well with fuchsin or methylene
blue. Brief staining in Giemsa's solution gives it a lavender color; with more
prolonged staining it becomes reddish. The general aspect of the organism in
stained smears (Figs. 1 and 2) resembles that of Bartonella bac~llformis in similar
preparations, but the individual organisms are more sharply defined than are theindividual bartonellas, owing perhaps to a greater affinity for the dyes. No tendency to bipolar staining has been noticed.
O n e of t h e s t r i k i n g f e a t u r e s of t h e o r g a n i s m is its c h a r a c t e r i s t i cspirally c u r v e d flagella (Fig. 4) w h i c h are p r e s e n t at one end of t h eb o d y a n d m a y be two, three, or four in n u m b e r . So far as I a maware, t h e o n l y o t h e r p a t h o g e n i c o r g a n i s m h a v i n g flagella of this t y p e
is Bartonella bacilliform{s.

mDEYONOGUCm167

Because of its presence in material derived from Peru, theorganism has been named Bacterium peruvianum.

Pathogenicity.
When B. peruvlanum is inoculated intradermally into monkeys(Macacus rhesus, Cercopithecus callitricus) or dogs, the sites ofinoculation become edematous, congested, and necrotic within a few
days, and open ulcers with raised margins finally result; these usually
heal within 10 to 14 days. The Strain P 5 was more actively pathogenic than the J 45. Unbroken nodules such as were occasionallyproduced b y the P 5 strain resolved after about 10 days.
Cultures made from the blood of the animals during the height of
the local reactions remained sterile in all instances; there was no invasion of the general circulation by the organism. Whether or not.
the injection of B. peruvianum into the blood circulation in large
quantities will result in fatal septicemia has not been determined.
B. peruvianum induces an acute fatal septicemia in rabbits, guinea
pigs, rats, and mice, when administered intravenously or intratesticularly. A broth culture 48 to 72 hours old was lethal within 24 to 48
hours in a quantity of 0.1 to 1 cc. Autopsy reveals very striking andcharacteristic changes in the abdominal viscera.
The liver seems to be most affected; it is somewhat enlarged, tense, uniformly
peppered with innumerable minute, intensely red spots which make the wholeorgan appear red. The stomach shows many diffuse and punctiform hemorrhages
recognizable through the congested serosa. The entire intestine appears diffusely
limpid red. The kidneys are swollen and congested, and the adrenals are much
enlarged and deep red. The spleen is not noticeably enlarged but is soft andextremely dark bluish red. The peritoneal cavity contains some limpid pink
fluid. No fibrinous exudate was found on the surface of any organ. The abdominal muscles are frequently spotted with hemorrhagic areas of moderate size.
The lungs are congested but show no hemorrhages. The heart is flabby, but there
is no pericarditis or Pleurisy. B. peru~ianumcan be recovered in pure culture
from heart's blood or spleen.
When inoculated intratesticularly, the animals succumb within 1 to
3 days and show the same changes as do animals injected intravenously. The scrotum and testicle both show intense edema, congestion, hemorrhages, and necrosis.

168

ETIOLOGYO~F O R O Y AFEVER.

IX

There was no intercurrent infection with B. lepisepticus or B. monocytogenes~ in any of the animals.

Differentiation of B. peruvianum from Other Pathogenic Microorganisms.
The characteristics of B. peruvianum may be summarized as follows:
Proper motility by means of multiple unipolar flagella; non-retentionof Gram's stain; strict aerobiosis; better growth at 25°C. than at37°C.; presence of proteolytic and absence of sugar-splitting ferment;
mucin production in broth; hemolytic action; and a wide range ofcharacteristic pathogenic properties for rabbits, guinea pigs, rats,mice, dogs, and monkeys. No microorganism having been previously
described which corresponds with the foregoing description, theorganism in question is regarded as a new species.
Bartonella bacilliformis resembles B. peruvianum in morphology,
in its inability to ferment carbohydrates, in growing better at 25°C.
than at a higher temperature, in not retaining Gram's stain, in being
strictly aerobic, and in having one to four unipolar flagella. The twoorganisms differ widely in other respects, however. Bartonella bacilliformis is unable to grow on any fluid medium so far tried, or any
medium which does not contain blood elements; it grows very slowly,
and the colonies are very minute; it lacks proteolytic or hemolytic
activity, it is difficult to disperse into a homogeneous suspension, andabove all, it has specific pathogenic properties. Moreover, a potent
anti-bacilliformis immune serum gives a specific complement fixation
and a distinct agglutination reaction with BartonelIa bacilliformis,
but none with B. peruvianum.
B. proteus and allied organisms are distinct from B. peruvianum inmorphological and cultural properties. The flagella of the proteus
group are peritrichal, and hydrogen sulfide is produced by the organisms in the course of growth.
The coIi-enteritidis-typhoid group ferments carbohydrates, and does
not liquefy L6ffler's serum. The flagella of this group are peritrichal.
2Murray, E. G. D., Webb, R. A., and Swalm, M. B. R., J. Path. and Bact.,
1926, xxix, 407. Pirie, J. H. It., Pub. South African Inst. Med. Research, 1927,iii, 185, I am indebted for cultures of this microorganism to Dr. J. C. G. Ledingham, Director of the National Collection of Type Cultures at the Lister
Institute.

HIDE¥O NOGUCHI

169

B. monocytogenes has a single polar flagellum and the property offermenting a great many carbohydrates.
Bacillus rickettsiformis 8 resembles Bacterium peruvianum in somerespects, in morphological features, in growing best at low temperatures, and in producing mucin in broth cultures. But the rickettsiformis is non-pathogenic and ferments several sugars.
SUMMARY.

A minute, pleomorphic, motile, Gram-negative bacterium has been
isolated from two specimens of nodular tissue from human verruga.
In films and sections of the original tissues the organism in question isdifficult to distinguish from Bartonella bacilliformis, with which it wasassociated, and even in pure culture it has a number of properties incommon with that parasite. No sugars are fermented by it, it is anobligate aerobe, the optimum temperature for its growth is 25°C., andit has two to four spiral flagella attached to one end of the body. Itis, however, readily cultivated on any ordinary culture medium.
Broth cultures contain much mucin, but no hydrogen sulfide is formed.
Coagulated serum is liquefied by its growth, and the red corpuscles ina blood agar plate are hemolyzed.
Rabbits, guinea pigs, rats, and mice develop acute, fatal septicemia asa result of intravenous or intratesticular inoculation of young cultures.
The liver is characteristically affected and shows a general parenchymatous degeneration and necrosis; the entire gastrointestinal tract isintensely congested, and numerous hemorrhagic areas are present; thespleen, dark and soft, is rarely much enlarged; the kidneys are swollenand congested; the adrenals are much swollen and intensdy red; thelungs are sometimes congested but otherwise normal. In the case of
intratesticular inoculation the scrotum and testicle both undergo
rapid gangrene. In monkeys no septicemia has been observed, but
a violent local reaction--swelling, congestion, sometimes necrosis-follows intradermal inoculation.
Since no microorganism corresponding in character with this one
has previously been described, it is regarded as a new species, andbecause of its presence in material obtained from Peru it has been given* Noguchi, H., Y. Exp. Med., 1926, xliii, 515.

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ETIOLOGY OF OROYA F E V E R . I X

the name Bacterium peruvianum. The significance of the associationof B. peruvianum with Bartonella bacilliformis deserves further investigation; it is not impossible that the two organisms are introduced
into the human body by the same blood-sucking insect.

THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X L V I I .

PLATE 6.

Cultures of Bacterium peruvianum, n. sp., an organism associated with Bartonellabacifliformis in verruga tissues.

FIG. 1.

FIG. 2.
Giemsa's stain.

FIG. 3.
Giemsa's stain.

× 1000.

Fro. 4.
X 1000.

Zettnow-Fontana flagella stain.

X 2000.

(Noguehi: Etiology of oroya fever. IX.)