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Journal of Experimental Medicine

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SPIROCH/ETA ICTEROHTEMORRHAGI/E IN AMERICANWILD RATS AND ITS RELATION TO THE
JAPANESE AND EUROPEAN STRAINS.

FIRST PAPER.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, January 23, 1917.)
Infectious jaundice has been known for a long time to occur among troops inbarracks, among sewer workers, miners, and rice planters, and its entity has beenrecognized (Larrey, Ozanam, 1849, Monneret, 1859, Laveran, 1865, Lancereau,
1882, Landouzy, 1882, Mathieu, 1886, and Weft, 1886). Weil described fourcases with typical symptoms, and the disease is very often called by his name.
Well's disease is characterized by sudden onset of malaise, often intense muscular
pains, high fever for several days, followed by jaundice and the appearance ofbile pigments, albumin, and casts in the urine; in severe cases epistaxis, subcutaneous hemorrhages, and lymphadenitis are observed.
A disease supposed to be identical with the Well's disease of Europe is prevalentin Japan, and in 1914 Inada and Ido succeeded in transmitting to guinea pigs the
typical experimental disease, accompanied by jaundice, hemorrhages, albuminuria, etc., by inoculating them with the blood of patients suffering from theJapanese form of infectious jaundice. In the blood and various organs of theanimals they discovered a new spirochete, which they also found in the humanspecimens. Spirockceta icterohceruorrhagiee, as it was designated by Inada andhis associates, z has since been proved beyond doubt to be the causative agent of
the disease in question. A year and a half later, Uhienimth and Fromme," andalso Hfibener and Reiter,3 showed that the Weil's disease prevalent among theGerman soldiers during the present war could also be transmitted to guinea pigs
by injecting them with the blood of patients. It was not until 1916, however,that Htibener and Reiter* announced the finding of a spirochete in the experimental animal material (liver, blood, kidney, etc.), which they called Spiroche~ta
z Inada, R., Ido, Y., Hold, R., Kaneko, R., and Ito, H., J. Exp. Med., 1916,
xxiii, 377.
Uhlenhuth and Fromme, Med. Klin., 1915, xi, 1202, 1264, 1296, 1375.
Hiibener and Reiter, Deutsch. reed. Woch., 1915, xli, 1275.
* Hiibener and Reiter, Deutsch. rued. Woch., 1916, xlii, 1.
755

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SPIROCH2ETA ICTEROH3EMORRttAGI3E

nodosa. Following the publication in America1 of the article by Inada and his
collaborators, Stokes and Ryle~succeeded in transmitting the disease to guinea pigs
by inoculating them with the blood of British soldiers in Flanders who had Well's
disease, confirming at the same time the presence of a spirochete closely resembling
the Spirockceta icterohcemorrhagi~e of the Japanese workers. Stokes, Ryle, andTytler 6 left the question of the identity of the two strains (the Belgian and theJapanese) open for future discussion. In the meanwhile, Martin and Pettit ~' s
Costa and Troisier, °' t0 Gamier and Reilly, n Renaux, TM Merklen and Lioust, TM
Ameuille, 14 Salomon and Neveu, TM and others, working among the French soldierson the western front, reported similar clinical and experimental findings. They
also considered the spirochete isolated from the French specimens to be closely
related to the Japanese strain. On the Italian front numerous cases of jaundice
have been observed, and Monti t6 has demonstrated the spirochete in the experimental material. According to MacKenzie, there were at one time a considerable number of cases of infectious jaundice among the Canadian soldiers stationed
at Salouica. 17 It is of interest to note that'while the mortality among the Japanese is as high as 38 per cent, that of the European soldiers did not exceed 2 to 3
per cent. It seems reasonable to assume that the Japanese strain has alreadyacquired a marked increase in virulence for human subjects, owing probably to amore frequent passage from man to man. Such transmission is more frequent inJapan, for example, among the rice planters and miners with imperfectly protected feet, than in Europe, where exposure to the infection was brought about
only through the unsanitary conditions of war.
T h e e n t r a n c e of this spirochete i n t o the h u m a n b o d y seems t o beof c o m p a r a t i v e l y recent occurrence. T h e d i s c o v e r y of t h e spirochetein a p p a r e n t l y h e a l t h y wild r a t s c a u g h t n e a r the infected districts
5 Stokes, A., and Ryle, J. A., J. Roy. Army Med. Corps, 1916, xxvii, 286.
s Stokes, A., Ryle, J. A., and Tytler, W. H., Lancet, 1917, i, 142.
7 Martin, L., and Pettit, A., Presse m~d., 1916, 569.
s Martin and Pettit, Bull. Acad. rn~d., 1916, lxxvi, 247.
s Costa, S., and Troisier, J., Compt. rend. Soc. biol., 1916, lxxix, 1038.
to Costa and Troisier, Bull. et ~u~m. H6p. Paris, 1916-17, xl, series 3, 1928.
n Gamier, M., and Reilly, J., Bull. et rn2m. H~p. Paris, 1916-17, xl, series 3,
2249.
Renaux, E., Compt. rend. Soc. biol., 1916, lxxix, 947.
is Merklen, P., and Lioust, C., Bull. et ~ m . H6p. Paris, 1916-17, xl, series 3,
1865.
14 Ameuille, P., Bull. et m~m. H~p. Paris, 1916-17, xl, series 3, 2281.
Salomon, M., and Neveu, R., Compt. rend. Soc. biol., 1917, lxxx, 272.
16 Monti, A., Boll. Soc. reed. chit. PaPa, 1916, Nos. 3-4.
1~ Personal communication.

HIDEYO NOGUCHI

757

seems to support the theory that the disease was originally epizootic
among certain rodents, particularly wild rats, and after a long sojourn in this species of hosts its virulence for these animals has been
reduced to such an extent as to cause the latter no inconvenience, or,at least, a state of tolerance for the spirochete has developed.
Inada, Ido, Hoki, Ito, and Wan118found nearly 30 per cent of all the rats examined to be infected; that is, to carry the spirochete in their kidneys. Stokes
Ryle, and Tytler,8 as well as Martin and Pettit, 19 were able to demonstrate thespirochete in rats captured on the western front, not only in those taken in theimmediate neighborhood of the infected zone, but also in rats captured in localities some distance from the trenches.
In America, especially the United States, there have been few epidemic or endemic cases of infectious jaundice reported from various
quarters of the continent (Toronto, Middle Western and Southern
United States) and from Cuba, but it was not known whether or notthese cases corresponded with those found in Europe and Asia. The
discovery of the specific pathogenic agent now enables us to answer
this question experimentally.

Isolation of the Organism.
We have collected a large number of wild rats in this country andremoved their kidneys for the purpose of ascertaining whether or not
the organs contained the spirochete which causes the typical experimental lesions characteristic of the organism of infectious jaundice.
Leaving tile experimental details for a future communication, it m a y
be well to state here briefly that b y inoculating the emulsion madeof the kidneys of forty-one wild rats into fifty-eight guinea pigs during the last 3 months, we have been able to produce in three groups ofguinea pigs (four in each group) a typical icterohemorrhagic spirochetosis altogether identical with the findings in the guinea pigs
which died of the injections of the Japanese and Belgian strains of
S pirochceta icterohcemorrhagice.
Since it was our practice to make an emulsion from the kidneys(eight) of four wild rats and inject the same into four to six normal
is Inada, R., Ido, Y., Hoki, R., Ito, H., and Wani, H., J. Exp. Med., 1916,
xxiv, 485.
19Martin and Pettit, Compt. rend. Soc. biol., 1917, Ixxx, 10.

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SPIROCIZ~TAICTEROII~MO~GL~

guinea pigs,it is difficult to say whether the icterohemorrhagic spirochetosis produced in some of the guinea pigs in each group was due
to one or more of the rats employed. It would have been better if
we had inoculated several guinea pigs with the emulsion of kidneys
from each rat, but this would have involved a large number of guinea
pigs, and hence our method of mixing four in each group. Since thesuccessful isolation of the organisms from three of the pooled groups,
we have resorted to individual tests, the results of which will be reported later.
The strain of spirochete isolated from the American wild rats caught
in the vicinity of New York City produced death ill guinea pigs within
9 to 12 days, attended by marked jaundice, cholemia, choluria, andextensive hemorrhages in various viscera. The temperature seldom
exceeds 39°C.; that is, it does not show the fever which usually precedes the collapse and the appearance of jaundice in the guinea pigs
inoculated with the Japanese or Belgian strains, which have at thistime been passed through many guinea pigs. The spirochetes arefound to be abundant in the internal organs, but are only moderately
numerous in the blood. In the urine of some guinea pigs, which succumbed to the experimental icterohemorrhagic spirochetosis, varying
numbers of the organisms were found in the urinary casts.
The morphology of the organism corresponds with that of theJapanese and Belgian strains, with which we were able to compare
it.s0 Its elementary structure is that of a closely wound slender
cylindrical thread with gradually tapering ends, averaging 9 by 0.25
v. Individuals of 3 to 4 ~ or 20, 30, and even 40 # are met with in a
culture. The number of coils is greater ill a given length than thatof any spirochete hitherto known. It is so closely wound that within
5 u there are 10 to 12 coils. Near the extremities, the coils become
closer. They are never very deep, and in general, the aspect of thewhole body is that of a transversely barred chain of streptococci.
The winding is rarely seen distinctly, although it can be brought out
well by a carefully fixed stained preparation (osmic vapor fixations0 1 am greatly indebted to Dr. Victor G. Heiser for the Japanese strain and to
Dr. Alexis Carrel for the Belgian strain. The Japanese strain was kindly furnished by Dr. M. Miyajima, and the Belgian by Dr. Carl Browning, who obtained
it from Dr. Adrian Stokes.

HII)XYO NOOVCat

759

and Giemsa stain), or under powerful dark-field illl~ralnation. I tshould be noted that the description of the organism by most authors
leaves this point.unclear, and so far no satisfactory microphotographhas been reproduced. The movement and customary position of theorganism in a free space are characteristic. Active specimens Showa straight body with one or both ends curved in the form of a semi-:
circle. The length of the hook at the end varies somewhat but is
usually about 3 to 5 #. While in motion, the organism, without re]axing its elementary minute windings, rotates around its axis, making about two to four turns per second, giving the impression of adrawn out figure eight. The movement is bipolar, and its direction
alternates at short intervals. When passing through a semisolid
medium, such as fibrin or soft agar, the body of the spirochete as'sumes a wavy spiral not unlike the refringens type. The number ofwaves m a y vary from a few broad ones to as many as five or six insome of the long individuals found in a culture. The movements
are brusque and erratic, changing suddenly the direction of progression. At times, the organism moves about with one end taking various directions. The facility with which these spirochetes travel
through the fibrin or semisolid agar medium is without a parallel
among any known spirochetes. The body is absolutely flexible.
There is a distinct halo around the organism, but no membrane has
so far been demonstrated. The part of the body which forms thehook terminates in a fine point, but no minute flagellum-like projection could be demonstrated by staining (Loeffier, Pitfield, CasaresGil, Fontana, etc.), or by dark-field illumination. It is devoid of aterminal filament such as is characteristic of a spironema or treponema,
and is resistant to saponin (10 per cent), unlike all other spirochetes.
It calls for a new genus, and on account of its fine and minute windings, the name Leptospira is suggested.
The hooked ends form one of the most characteristic poses of theorganism while rotating on its axis in a free space, but as soon as itmeets a solid or semisolid obstacle, it begins to penetrate into it.
Its habitat seems to be a porous gelatinous mass of substance, theorganisms swarming in and out of it. In a culture the majority ofthe organisms will be found in a semisolid piece of medium. After
death it may retain its position at the moment of death; hooked or

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SPIROCI-I~TA ICTEROH2EMOR_R.H.AGI~

contorted forms, resembling the letters c, s, 1, and b, are most frequently met with among immobile or dead organisms. W h e n destroyed b y a strong acid or alkali, t h e y lose their minute curves,swell up, and become indistinct and straight. T h e organism has t h epower of almost perpetual motion. A culture as old as 3 m o n t h s
still displays as m u c h vigor as does a young one. None of the othervarieties of spirochetes have this p r o p e r t y in cultures. T h e organism
passes the Berkefeld candle V.
Cultures.
The Japanese investigatorst succeeded in obtaining a culture of their strains byusing a medium similar to that previously introduced by the writer for the spirochete of relapsing fever.21 Ito and Matsuzaki approved this technique and recommended also the use of blood gelatin or blood agar in various concentrations.~
In Europe, Reiter~ cultivated the German strain in the sera of various animals,
sometimes diluted several times with isotonic salt solution. Recently, Martin,
Pettit, and Vaudremer~ obtained a culture of the French strain by the use of:animal sera, particularly that of beef, diluted with Locke's solution. In all these
instances, the surface of the culture medium was covered with a layer of sterile
paraffin oil, as recommended by the writer for the cultivation of spirochetes in:general. The Belgian strain of Stokes has not yet been cultivated, and Stokes,
Ryle, and Tytler particularly laid stress on its resistance to artificial cultivation
as a characteristic of the strain.
T h e writer has been interested in determining the cultural conditions which will give uniform results, and has found t h a t the following
methods give the most successful growth, not only with the Japanese,b u t also with the Belgian (Stokes) and American strains, which wereobtained in p e r m a n e n t cultures for the first time b y these means.
I n our experience, it seemed best to m a k e a distinction betweent h e initial culture or the first generation and the subcultures, since
there is a great difference in the readiness with which the first a n dsubsequent generations of the spirochetes grow in culture. F o r example, all three strains, in our hands, failed to grow, or grew poorly
sl Noguchi, H., J. Exp. Med., 1912, xvi, 199.
~2Ito, T., and Matsuzaki, H., ]. Exp. Med., 1916, xxiii, 557.
z~ Reiter, Deutsch. reed. Woch., 1916, xlfi, 1282.
Martin, L., Pettit, A., and Vaudremer, A., Compt. rend. Soc. biol., 1917,
lxxx, 197.

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761

in the various media recommended by different authors. The bestprocedure, and one which has always been reliable for securing initial
growth was to produce strands of loose fibrin in the fluid culture media
by using a small quantity of citrate plasma in combination with thediluted or undiluted serum of a suitable animal. The beneficial effect of a loose fibrin upon the culture of the spirochetes of relapsing
fever has already been mentioned by the writer, st The dilution of theserum m a y be made in any proportion above 1 : 10 by adding a sterile
saline (0.9 per cent) Ringer solution, or even plain water. For obtaining the spirochetal material for inoculation, the citrate blood derived from the heart of a guinea pig having the disease is best, although an emulsion of the liver or kidney may also be used. Whenno secondary bacteria are present, a positive culture can be secured
at the first attempt. By using graduated quantities of the infectedguinea pig's blood (citrate), the writer secured a good growth in as
high a dilution as 1 : 100,000.
Undoubtedly blood cultures for diagnostic purposes in humancases are feasible with a suitable medium. For this purpose I should
recommend two different media, one being apparently as good as
the other: (a) rabbit serum 1 part + Ringer or 0.9 per cent sodium
chloride solution 3 parts + citrate rabbit plasma 0.5 part, covered
with a thin layer of sterile paraffin oil; (b) the same, except for theuse of 0.5 to 1.0 part of neutral or slightly alkaline agar (2 per cent),
which should be added while in a liquid state and quite hot (6065°C.) in order to get a uniform mixture of the agar. These culture
media, because of the paraffin oil layer, can be preserved at room temperature for many months, though a cooler place is better. They
may be inoculated with suspected blood by introducing a quantity
which is regarded as adequate in each instance. In the case of an
infected guinea pig, the detection of the spirochete in the blood canbe made within 48 to 72 hours, if the culture tubes are placed at 3037°C. The search for the organism should be made within the aerobic zone immediately below the surface (1.0 to 1.5 cm.), because, according to the experience of the writer, the organism is an obligatory
aerobe, unable to grow in the absence of oxygen. This would explain
the unsuitability of a solid medium, which prevents access of air.
The use of fresh tissue, as in the case of the relapsing fever spiro-

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SPIROCH~TA

ICTEROH/EMORRHAGI3E

chete, is not required for the cultivation of this organism, and is perhaps seriously detrimental in a fluid medium. Growth takes placeat any temperature from 10° to 37°C., being much more rapid at
higher than at lower temperatures. The growth is almost invisible
in a fluid medium, but appears as a somewhat distinct haze in semisolid medium such as described above (a and b). The appearance
is not unlike the hazy, diffuse growth of various treponemata in similar media, the only difference being that the haze in the case of thelatter stops abruptly a few cc. below the surface, while the present
organism is unable to grow beyond a few cc. below the surface. So
far, no investigator has mentioned the visible growth of the spirochete, all describing it as invisible.

Virulence.
The American strains possess an average degree of virulence, having
killed guinea pigs in 9 to 12 days in the first generation in this animal.
The lesions, as well as the jaundice, were severe and typical in every
respect. The second passage killed the guinea pigs in a much shorter
period, 6 to 8 days, the lesions being similar to those of the first passage. Again there was no fever. They seem to be running a course
similar to that of the Japanese strain in attaining a higher virulenceby successive passages. The virulence of the Belgian strain appears
to be less readily increased, as the guinea pigs survive 9 to 10 days.
This distinction in virulence may be due to the fact that the Belgianstrain was brought to us in infected rats, instead of in guinea pigs,
in which the Japanese strain was received.

Immunity Relations.
Reserving the experimental details for a fuller report, it may bestated briefly that the guinea pigs which had been made immune to
the Japanese or Belgian strain resisted the inoculation of the liveremulsion containing large numbers of the spirochetes of American
origin, while control animals succumbed to the typical infection in
6 to 8 d a y s . The blood sera from guinea pigs and rabbits immunizedwith the Japanese or Belgian strain agglutinated the American strain
as strongly as they did their own strains. The immune sera exerted

~mEYo NOOUCHI

763

germicidal or lyric action upon the three strains indiscriminately.
Whether or not protection experiments in guinea pigs will show any
distinctions between t h e Belgian and the Japanese strains on the one
hand and the American on the other will shortly be determined.
As far as the former are concerned, the active immunity developed inguinea pigs or rabbits and their immune sera are effective, the one
against the other, in almost equal titers.
The finding of the causative organism of infectious jaundice amongwild rats in America, and the identification of this strain with thosefound in Asia and Europe seem to be particularly important in revealing a latent danger to which we have been constantly exposed
but from which we escape as long as sanitary conditions are not disturbed by untoward events.
S~&RY.

The principal points brought out in the present article are thefollowing.
1. Wild rats captured in this country carry in their kidneys a spirochete which possesses the morphological and pathogenic properties
characteristic of Spiroclueta icterohcemorrhagieediscovered by Inada in
the Japanese form of infectious jaundice.
2. Cultures of the American, Belgian, and Japanese strains of the
spirochete were obtained by a special technique described, the firsttwo strains having been cultivated artificially for the first time.
3. Animals actively immunized against the Japanese strain resist
inoculation, not only of the same strain, but also of the Belgian andAmerican strains. The Belgian strain produces immunity equallyeffective against all three strains. Experiments to ascertain whether
the immunity afforded by' the American strain also protects against
the Japanese and Belgian strains are in progress.
4. These findings warrant the conclusion that the spirochetes designated here as the Japanese, Belgian, and American strains are probably identical.
5. On account of its distinctive features, a new genus, Leptospira,has been suggested as the designation of this organism.