野口英世著

Journal of Experimental Medicine

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C U L T I V A T I O N OF S P I R O C H 3 E T A G A L L I N A R U M . *BY H I D E Y O NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)
PLATE 68.

Spirochcta gallinarum, discovered in 19o 3 by Marchoux andSalimbeni 1 in an epidemic in Brazil, is a septicemic organism responsible for a febrile disease of the chicken which often terminatesfatally. The infection is transmitted by certain species of ticks(.drgas miniatus, Argas persicus, Ar.qas reflexus, Ornithodorus
moubata), and the symptoms appear usually within three or fourdays after the tick's bite, and last for severa.1 days; there is no
relapse. At the height of the fever the blood swarms with the
spiroch~et~e, but in the event of recovery, they disappear withinseveral days. Spirochwta gallinarum bears resemblance to Spirochwta anserina, discovered by Sakharoff 2 in the blood of geese anda spiroch~eta found by Balfour 8 in a febrile chicken disease in
Sudan, and Nuttall considers that the three forms are probably
identical. According to Sakharoff, the anserina is incapable of infecting the chicken, while the gallinarum can infect not only thechicken, but sparrows, geese, and rabbits.
Spirochcetagallinarum bears a close morphological resemblance tospiroch~et~e causing relapsing fevers in man, but is somewhat moredelicate than they. According to Novy and Knapp, 4 the gallinarum
measures about 0.2 5 of a micron in width and from IO to 12 microns in length. In a fresh preparation of blood from infected birds
it exhibits remarkable motility characterized by a swift forward
movement and a lateral vibration of the entire body. It often forms
a ring, turning first in one direction, then in the reverse direction.
* Received for publication, August I5, 1912.
1 Marchoux, E., and Salirnbeni, A., Ann, de l'Inst. Pasteur, 19o3, xvii, 569.
Sakharoff, M. N., ./Inn. de l'Inst. Pasteur, 1891, v, 564.
a Balfour, Reports of The Wellcome Research Laboratories, 19o8, iii, 38.
* Novy, F. G., and Knapp, R. E., Jour. Infect. Dis., 19o6, iii, 291.

620

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In some specimens when one end of the body is attached to theslide a swinging movement may be observed.
Spirochceta gallinarum has not previously been successfully cultivated outside the body. Borrel and Burnet 5 observed an initial
multiplication of the organism when a portion of the infected blood
was mixed with chicken plasma, either pure or citrated, to whichsome bouillon was added afterwards; but no multiplication took
place beyond the first tube. Levaditi 6 observed multiplication formany generations by cultivating the spiroch~eta~ in the peritoneal
cavity of rabbits in collodion sacs containing infected blood.
in the course of my studies on the cultivation of spiroch~et~e ofrelapsing fevers, ~ I undertook to cultivate in vitro Spirochceta gallinarum,, and with success. I have now secured many generations bypassing the culture from tube to tube at appropriate intervals.
MateriaL--Two different strains of Spirochwta gallinarum were
employed. The first strain was kindly furnished by Professor Ehrlich in infected ticks from which the spiroch~eta was subsequently
transferred to chickens. The infection of chickens by the ticks
readily succeeded, The second strain of the gallinarum I owe to thecourtesy of Dr. Levaditi of the Pasteur Institute o f Paris. It wassent in infected blood hermetically sealed in glass tubes. Upon
arrival, fifteen days later, a few motile spirocha~ta~ were still seen
under the dark-field microscope. The blood was first inoculated
into canary birds and from the canary to the chicken.
For the purpose of cultivation the blood was aseptically taken
under ether anesthesia from the heart of the infected birds andmixed immediately with a sufficient quantity of sodium citrate salt
solution to prevent coagulation. The resulting emulsion of theblood which contained varying numbers of the spiroch~et~e was used
for inoculating the culture media.
Culture Media.--The essential constituents of the culture media
are: ( I ) f r e s h tissue, (2) ascitic fluid, and (3) paraffin oil. It is
understood that the tissue, ascitic fluid, and paraffin oil must be absolutely sterile. The tissue must be perfectly fresh and of proper size.
5Borrel, A., and Burner, E., Compt. rend. Soc. de biol., I9o6, Ix, 540.
*Levaditi, C., Compt. rend. Soc. de biol., 19o6, Ix, 688.
'~Noguchi, H., Your. Exper. Med., 1912, xvi, 199.

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Cultivation of Spiroch~ta gallinarum.

Kidneys of normal rabbits or pectoral muscles of chicken are b,stsuited for this purpose. The ascitic fluid must be tested for its
suitability, as many specimens are wholly unsuitable. The selection is to be made by actual cultivation experiment, which is donewith several specimens of ascitic fluids simultaneously. This may
seem very tedious, but when one finds good specimens, they can be
kept on ice for future work and there will be no difficulty in continuing the cultivation for any number of transfers. The paraffin oil
is best sterilized twice in an autoclave.
Technique of Cultivation.--Cultivation of this organism is simple
and consists in placing a piece of fresh sterile tissue (about the sizeof a chestnut) and ascitic fluid in a sterile test-tube, followed by the
introduction of several drops of the blood emulsion containing the
spiroch~et~e. A layer of sterile paraffin oil is now poured in. The
amount of ascitic fluid in the culture tube should be sufficient toreach a height of about ten centimeters. Incubation is at 37 ° C.
Mindful of the frequent occurrence of bacterial contamination,
it is my custom to inoculate more than one culture tube on eachoccasion in order to obtain at least one pure culture. Bacterial
contamination is a serious obstacle in the work, as when it occurs,
it cannot be removed without passing the culture once more into
chickens; sometimes the growth of the culture may be entirely suppressed according to the nature of the contaminating organisms.
Properties of Pure Culture.mSpirochcta gallina/rum increases
considerably in number within twenty-four hours of inoculation into
the culture media. The rate of multiplication continues to be very
rapid during the following three days, at which time several spiroch~et~e are found in each microscopic field. On the fifth day thegrowth comes to a standstill, and on or after the sixth day degenerative processes, indicated by reduction or loss of motility of manyorganisms and the irregularity of their curves, commence to prevail.
While the majority of the spirochaetal organisms undergo disintegration within ten days, a few motile ones are still present in the cultures kept at 37 ° C. on the fifteenth day, and very rarely is a living
specimen present in cultures older than three weeks, s The onset ofs A n exception to this rule has recently been met with while examining some
culture tubes which bad not been disturbed since inoculation thirty days previous'ly and which had been continuously kept in the thermostat at 37 ° C. T h e r ewere still quite a few motile spirochaet~e.

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623

the degenerative phase takes place gradually and is in striking contrast to the suddenness wi,th which it occurs in the cultures of
Spirochtet¢ duttoni, kochi, obermeieri, and novyi.
The appearance of the culture media is but little modified by thegrowth of Spirochceta galIinarum, except that under certain conditions a very faint opalescence may appear. This happens especially when a piece of pectoral muscle of the chicken is used insteadof rabbit kidney. No noticeable odor is produced in the culture.
No growth is vbtained at room temperature or in vacuo at 37 ° C.
Thus it is clear that the organism.requires for its growth a certain
amount of oxygen. In this respect the gallinarum resembles the
spiroch~etm of human relapsing fevers.
Subculture,C.--Tran.splantation from culture to culture can be
kept up for many generations and probably for indefinite periods,
provided that a suitable specimen of ascitic fluid be employed. I
have been able to continue cultivation of the two strains I worked
with for fifteen successive transfers during the past two months.
In continuous cultivation, the importance of suitable ascitic fluids
cannot be too much emphasized.
Morphology.--(Figures I to 12.) In culture media containing apiece of fresh muscle of chicken and ascitic fluid, Spirochceta gallinarum presents all the morphological characteristics observed in the
blood of an infected chicken. The organism measures about 0. 3 ofa micron in width and from 8 ¢o 16 microns in length. Cultures
three or four days ,old show numerous chains of two, three, or evenfour organisms. The organisms may be joined together by a short,thin filament that measures less than two microns in length, butsometimes the joint is merely suggested, from the absence of curves,and there is no actual thinning. The number of curves varies according to the length of the specimen, but the distance between the
curves is constant, measuring about 1.8 microns. Average curves
have a depth of about one micron and usually a round apex. The
position of the curves appears to change especially when the spiroch~et~e manifest violen¢ movements. The regularity of the curves ismost marked in the specimens which show moderate motility. 9 The*The regularity of curves may be more or less lost during the process of drying on a slide, so that the stained preparation shows rather irregularly curved
spiroch~et~e (figure 12).

624

Cultivation of Spirochceta gallinarum.

movements are chiefly rotatory with more or less marked lateral
vibration. The forward locomotion is less noticeable in the culture
than in the blood. Agglomeration of spiroch~et~e is seldom observed even at the height of growth (usually on the fifth day) andin this respect the condition of culture differs from that of the bloodof an infected bird.
Spirochceta gallinarum usually possesses a delicate, finely curved
projection attached to one end of the body, measuring about three
microns. It is rare to find a specimen with similar projections at
both ends. In some specimens the projection is devoid of curves
and appears to be more fragile than the curved ones. I am of theopinion that the straight projection is a prolongation of periblastand the curved one is continuous with the axial filament of the
spiroch~eta. I arrived at this conclusion from my observations on
cultured Treponemata (including the paUidum, microdentium, macrodentium, mucosum, refringens, and other relapsing fever spiroch0et~e).
Under somewhat less favorable cul.tural conditions certain atypical forms arise. These organisms may be short, having only twoor three curves, or the curves may be irregular. This irregularity
may also arise when a less suitable ascitic fluid is used and in a culture entering on the degenerative phase.
In all cultures one invariably finds varying numbers of roundrefractory bodies that are attached to the periblast of the spiroch~et~e.
As a rule, one organism carries .one such body which may be attached to a point anywhere along the side of the spiroch~eta. This
spherical body measures about o.75 of a micron. The number ofthe organisms with round bodies is inconstant in different cultures,but it seems to become greater where the growth of the culture approaches its maximum. Usually it is absent in cultures not older
than two days. Apparently not every culture is capable of producing these bodies. Similar round bodies have been observed byprevious investigators not only in the blood of the infected birds,
but also in the fresh preparations of various other spiroch~etm. I
have described the presence of such round bodies in pure culturesof Treponema paIlidum, Treponema microdentium, Treponema mac-rodentium, Treponema mucosum, 1° Spirochceta refringens , Spiro10Noguchi, H., .four. Exper. Med., 1912, xvi, I94.

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625

chwta phagedenis, 11 Spirochceta duttoni, Spirochceta kochi, Spirochceta obermeieri, and Spirochceta novyi. The conditions underwhich the bodies are formed suggest that they represent one of thephases of the life cycle and should not be viewed as the result ofplasmolysis, as some investigators have assumed. 12 At the sametime they are certainly not the encystment forms described byPerrin, 13 von Prowazek, 14 and others in certain spiroch~etm.
Another phenomenon observed in pure cultures of Spirochceta
gallinarum, although common with all other spiroch~et~e studied
by me in culture, is the formation of numerous granules in certain
cultures, especially when their maximum growth has passed or the
media have been somewhat defective. These granules are round or
ovoid and are highly refractory. They do not exceed 0. 3 of a
micron and usually are about 0.2 of a micron in diameter. Theyshow active molecular movements. They have doubtless been derived from the spiroch~et~e, since they are attached to numerousorganisms along the entire body length. These granular spirocha~tm may be quite actively motile or may show no sign of life. W h e n
the cultures are examined carefully, the granular spiroch~et~e areobserved to be actually undergoing degeneration, o n examinations day by day, these spiroch~et~e are seen gradually to give up the
granules until they become completely or almost completely denuded, their immobile skeletal axial filaments alone remaining. In
my opinion, the entire process is one of the degeneration, and the
granules are nothing but the fragments ,of the periblast of the
spiroch~et~e. Whether or not this phenomenon has any relation to
the formation of coccoid bodies described by Balfour, 15 Leishman, 16
Bosanquet, 17 Hindle, is and others in blood specimens, I cannot say.
At all events, the granules just described in cultures are incapable of
11Noguchi, H., ]our. Exper. Med., 1912, xvi, 261.
12Hindle, E., Parasitology, x912, iv, 463.
Perrin, W. S., Arch. f. Protistenk., 19o6, xii, I31.
1,yon Prowazek, S., Arb. a. d. k. Gsndhtsamte, I9o6, xxiii, 554; Mern. do Inst.
Oswaldo Cruz, I9O9, i, 79.
15Balfour, A., tour. Trop. Med., I91I, xiv, II3.
16Leishman, W. B., Lancet, 191o, i, iI.
1, Bosanquet, W. C., Quart. Jour. Micr. Sc., 191I, lvi, 387.
18Hindle, E., loc. cit.

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Cultivation of Spirochc~ta gallinarum.

causing the infection in chickens or of being cultivated in the media
described.
While the morphological features of Spirochwta gallinarum in
culture media containing chicken tissue remain typical for many
generations, the organisms cultivated in media with rabbit tissue
become somewhat thicker and the curves appear shallower. By
transplanting these organisms back to the chicken media they once
more become typical. On the other hand, the spirochmta grows
just as abundantly in the presence of rabbit tissue as it does in
the presence of chicken muscle.
In regard to the mode of multiplication I was able to observe
unmistakable instances of transverse division by strangulation, but
no longitudinal division could be detected. Whether or not thelatter mode of division does not occur also under certain conditions
must be determined later.
At no period of cultivation did bacillary forms appear, such as
are described by Hindle 19 as one of the phases of the life cycle of thisorganism in the tick.
Filterability.--In view of the findings of Novy and Knapp, 2° who
caused an infection by injecting the filtrate of the blood derived
from animals int~ected with Spirochwta novyi, I made several seriesof experiments in order to determine whether the cultures of Spirochxta galliuarum contained at any period a filterable form. For
this purpose a mixture of cultures of varying ages (from two tothirty days) was filtered through several Berkefeld filters (V).
The filtrate was tested in two ways. It was inoculated into chickens
and cultured in suitable media. Controls for both series were provided with unfiltered cultures. Neither infection of chickens nor
culture was obtained with the filtrates. In this connection I may
mention that similar experiments performed with cultures of Spirochceta duttoni, kochi, obermeieri, and novyi gave negative results.
These experiments do not affect the positive results obtained by
Novy and Knapp, as they used filters which had been ground tolessen the thickness of the wall.
Pathogenicity.--Spirochceta gallinarum in culture does not lose
1,Hindle, E., loc. cit.
Novy, F. G., and Knapp, R. E., loc. clt.

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627

its virulence for the chicken to any noticeable degree when the transplantations are kept up at regular intervals in a suitable medium.
Thus the cultures after thirteen transplantations caused fatal infection in chickens with characteristic symptoms of the disease. I,tremains to be determined by future experiments whether or not
prolonged cultivation in artificial media will finally bring about an
attenuation or abolition of virulence of this organism. I havefound incidentally that when the transplantations into new media
are neglected for about four weeks a culture which still shows
numerous motile organisms no longer causes the infection in thechicken, even where a large quantity of the culture is inoculated. 21
This seems to indicate that the gallinarum may become attenuatedin virulence under certain cultural conditions; perhaps the attenuated cultures may confer immunity.
CONCLUSIONS.

I. Spirochwta gallinarum can be cultivated in suitable artificial
media for many successive generations and probably for indefinite
periods. The presence of fresh tissue and a certain amount ofoxygen seems ,to be essential for its growth. No perceptible odor is
produced in the cultures.
2. The maximum growth of Spirochwta gallinarum is reached
on about the fifth day, but the phase of degeneration commences
slowly and gradually, so that in this respect the gallinarum differsfrom the duttoni, kochi, obermeieri, or novyi, whose cultures areCharacterized by sudden onset of degeneration soon after the
maximum growth is attained.
3. No rod formation resembling bacilli arises in the course ofmultiplication of Spiroch(~ta gallinarum in cultures. Many roundor oval bodies appear in o}d cultures, but no infection of animals orformation of spiral forms from these granules has been produced.
The granules are probably the degeneration products derived fromthe periblast of the spiroch~et~e.
4- Cultures of Spirochwta gallinarum, either old or young, do
21The chickens which received the inoculation of the attenuated culture
resisted the infection against a virulent strain directly from an infected birdwhen tested two weeks afterwards.

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Cultivation of Spirocho~ta gallinarum.

not contain a f o r m which passes t h r o u g h a B e r k e f e l d filter ( V ) that
infects chickens or g r o w s into spiroch~et~e.
5. Spirochceta gallinarum r e m a i n s virulent f o r chickens a f t e r
being in cultures f o r at least thirteen generations, but it m a y become
avirulent u n d e r certain cultural conditions. T h e inoculation o fchickens with the a t t e n u a t e d culture renders the birds r e f r a c t o r y to
the subsequent infection with a virulent strain.
6. W h e n the spirochaet~e are cultivated in the m e d i a containing
rabbit kidney instead o f chicken muscle, the individual specimens are
s o m e w h a t thicker, but o t h e r w i s e typical.
7. Spirochceta gallinarum multiplies in culture b y transverse division. N o positive evidence of a longitudinal division has beenobtained.
EXPLANATION OF PLATE 68.
FIGS. I to IO. Schematic reproductions of Spirochcta gallinarum in pure culture, as observed under the dark-field microscope. Different stages of growthof the spirochaetm in a medium containing ascitic fluid and chicken muscle.
FIG. I. Average forms; FIG. 2, long chains; FIG. 3, short forms; FIG. 4,
ring formation; FIG. 5, agglomeration and knot formation; FIG. 6, doubled up
forms; FIG. 7, spiroch~etm with spore-like spherical bodies; FIG. 8, a spiroch~eta
with granules; FIG. 9, degeneration phase with numerous granules, skeletal axial
filaments, and several short motile spirochaetm.
FIG. IO. Spirocheeta gallinarum in a culture medium containing ascitic fluid
and rabbit kidney, showing somewhat thicker and less regularly curved forms.
FIG. II. Spirochcta gallinarum from a pure culture (2oth generation).
>( I,IOO. Dark-field view.
Fro. 12. Spirochaeta gallinarum from a pure culture (2oth generation) fixed
in methyl alcohol and stained with the Giemsa solution for four hours. X I,IOO.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XVI.

PLATE 68.