野口英世著

Journal of Experimental Medicine

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LOCALIMMUNITY TO TETANUS
RATS TREATED WITH

IN I N O C U L A T E DEOSIN.

BY HIDEYO NOGUCHI, M.D.
(From the Rockefeller Institute for Medical Research, New York.)

Previous studies having shown the mechanism of the immunity
to tetanus which develops in rats treated locally with eosin, my attention was directed by Dr. Flexner to certain indications of theoccurrence in the treated animals of a local in contrast to a general
immunity to the tetanus bacillus. Our studies 1 had already shownthat the spores on silk threads in the healed eosin-treated wounds
remain alive for an indefinite period, and the threads removed andtransplanted to other rats, or to the opposite side of the body ofthe same rat, caused fatal tetanus. And yet, the operation for theremoval of part of the spore-threads was not followed, in spite of
the trauma inflicted and the portion of the spore-thread which remained, by any tentanus in the extremity originally infected. Since
it appeared that the conditions were, after the second operation,
similar on both sides of the body, or in the two animals, an explanation of the different reactions observed was required. It seemednatural to assume, for the sake of further experiment, that therestricted germination and vegetation which take place in the eosintreated animals produce a small quantity of tetanus-toxin, which,acting locally upon the tissues, gives rise in them to a degree ofimmunity to the action of the tetanus poison. 2The observations upon which this hypothesis was first based will
be given ; but it should be stated in advance that the tetanus-bearing
threads were introduced subcutaneously in the thigh and not deeply
into the tissues. After the healing process is complete, the threads
come to lie in a small mass of scar tissue between the superficial
i Flexner and Noguchi, Your. o[ Exper. Med., I9o6, viii, i.
• Noguchi, "The Nature of the Antitetanic Action of Eosin," this number ofthe JOURNAL.
291

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Immunity to Tetanus in Inoculated Rats.

fascia and the skin. Except for the cicatrix the tissues all returnapparently to the normal condition.
Experiment / . - - I n order to determine whether the spores remained alive in
the inoculated rats after the healing process was complete, a portion of a thread
was removed thirty days after inoculation and fifteen days after the cessationof the eosin treatment; no trace of the eosin remained at this time in the tissues.
The removed portion of the thread was used for preparing cultures and for inoculating rat No. I2. Tetanus bacilli grew in the cultures. Tetanus developed inrat No. i2 on the second day, and death from tetanus took place on the fourth
day after inoculation. The treated and operated rat remained well.

Before discussing further the significance of this experiment,a selected series of protocols bearing on this question of local immunity to tetanus will be first given.
Experiment I[. Rat No. z3.--Thirty days after inoculation and fifteen days
after cessation of eosin treatment, a portion of the healed-in spore-thread wasremoved from the right thigh. No tetanus followed the trauma, and thewound quickly healed. Six days later, a second portion of the thread wasremoved and implanted subcutaneously under the skin of the left thigh. Tetanus
developed on the left (new) side on the second day, and death occurred on thefourth day after this inoculation. The right (original) hind leg remained
entirely free from tetanus.
Experiment Ili. Rat No. z4.--Eosin treatment. Seventeen days after inoculation the thread was removed from the right t h i g h ; the wound of the second
operation was completely healed in seven days, during which period no tetanus
developed. July i2, reinoculation of spore-threads beneath the skin of boththighs. July I6, left leg tetanic; right leg free. July I8, tetanus has progressedon left side; right side free. July I9, rat dead. The animal lived three days
after the first appearance of tetanus. The right (old inoculated) leg remained
free from tetanus until the end.
Experiment 1U. Rat No. r5,--Spore-thread inoculation into right leg June 8;
eosin treatment until June 20. No tetanus. Spore-thread removed July 28; notetanus. October 9 (I23 days after inoculation, and H I days after last injectionof eosin), 0.000o8 c.c. tetanus toxin injected into each thigh (0.0003 ~ m.l.d, inthree days). Tetanus developed in the left leg in forty hours, and advanced rapidly; slight tetanus developed in the right leg after three days~ and advancedslowly. Death on seventh day.
Experiment U. Rat No. 16.--Spore-thread inocuhition into right leg October
29; eosin rein treatment until November 8. No tetanus. December I (33 days
after inoculation and 25 days after last injection of eosin) 0.0004 c.c. tetanus
toxin injected into each thigh. Tetanus developed in left leg after three days,' and advanced rapidly; a mild form of tetanus developed in the right leg andadvanced slowly. Both sides became strongly contracted; death on sixteenth day.
Experiment VI. Rat No. ZT.--Spore-thread inoculation into right thigh October 19. Eosin gelb injection until November 2. No tetanus developed. December t (43 days after inoculation and 29 days after last injection of eosin),Hideyo Noguchi.

293o.ooo4 e.e. of tetanus toxin injected into each thigh. No tetanus developed in fivedays. Second injection into each thigh of o.ooo4 c.c. toxin. Next day tetanus
began in left leg; none in right leg. The tetanus advanced in the left andremained absent from the right side until death, which took place on lhe ninth day.
E x p e r i m e n t V I brings out clearly, for the first time, the existence of a degree of general i m m u n i t y to the toxin which is, however, able to off-set a small and limited a m o u n t only of the effectsof the toxin. T h e local i m m u n i t y of the right thigh proved, upona second injection of the toxin, to be still effective, while the general
immunity, t h r o u g h which the left leg had been protected f r o m theconsequences of the first injection, was exhausted by it. T h e n e x t
experiment indicates that even a higher degree of general immunitym a y arise f r o m the local infection.
Experiment VII. Rat No. /&--Spore-thread inoculation into right thigh;eosin treatment. No tetanus developed. October 9 (I23 days after inoculationand HI days after last injection of easin), o.oooo8 c.c. tetanus toxin (o.oo(x)8~m.l.d.) injected into each thigh. No tetanus after three days. October I2,0.o00o4 c.c. toxin injected into each leg. October I9, no tetanus developed. The
spore-thread from this animal transplanted to another rat produced typicaltetanic symptoms, followed by death in two and a half days.

E x p e r i m e n t V I I is a stronger confirmation even than experiment V I of the idea that general i m m u n i t y to tetanus toxin m a y
be developed in the eosin-treated rats. T h e degree of generali m m u n i t y is, in almost all the rats, lower than that of the localimmunity. Rat No. 18 seems to supply the one exception to what
appeared as the rule in this respect. It was now desirable to ascertain by a direct e x p e r i m e n t w h e t h e r the blood of the eosin-treatedrats contained a measurable quantity of tetanus antitoxin.
T h r e e series of rats were taken: ( I ) normal rats as controls;(2) rats which a f t e r the spore-thread insertion had been treatedwith eosin " g e l b ; " ( 3 ) rats which a f t e r inoculation had beentreated with eosin " r e i n . " T h e rats were bled from the carotid
artery, and the serum collected. On account of the small a m o u n tof blood yielded by these animals, the blood of the several animalsof each series was collected together. T h e eosin-treated rats werebled from s i x t y - f o u r to s e v e n t y - f o u r days a f t e r the cessation o f
the eosin treatment. T h e following tabulation gives the results ofthe tests. In each instance, the mixtures of serum and tetanus

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Immunity to Tetanus in Inoculated Rats.

toxin were kept at 37 ° C. for half an hour before injection.
injections were made beneath the skin of the thigh.
Serum Injected.

Toxin Injected.

Normal 0. 3 c.c.
Normal o. 3 c.c.
Saline o. 3 c.c.
Saline o. 3 c.c.
Eosin gelb " a " o. 3 c.c.

0.006 c.c. (6 m.l.d. )0.oo2 c.c. (2 m.l.d.)
0.006 c.c. (6 m.l.d. )0.002 c.c. (2 m.l.d.)0.oo6 c.c. (6 m.l.d.)

Eosin gelb " b " 0. 3 c.c.

0.006 c.c. (6 m.l.d.)

Eosin rein o. 3 c.c.
Eosin rein o. 3 c.c.

0.006 c.c. (6 m.l.d.)
0.002 c.c. (2 m.l.d.)

Tetanus Developed.

TheResult.

24 hours.
Death on 3 d day.
Death on 6th day.
24 hours.
Death on 3d day.
24 hours.
24 hours.
Death on 5th day.
slightly in 48 hours. Tetanus increased but
did not extend beyond
injected leg. 28 days
after injection still
alive ; tetanus gradually disappearing.
slightly in 48 hours. Tetanus progressed,
death on 9th day.
slightly in 48 hours. Death on 1oth day.
Death on ISth day.
5th day.

The experiments which I have set down in this article seem to
me to afford convincing proof of the existence of local immunity
to tetanus infection and tetanus intoxication. The observation
is, I believe, new and not without interest in its bearing on the theoryof the " building sites " of immune bodies in the animal organism.
Examinations of the lesion consisting of the scar enclosing thespore-threads have shown it to be superficial, limited to the fascia
and adjacent subcutaneous tissue, and to measure a few millimetersin extent only. There are no anatomical changes discoverable
which would connect the lesion with the deeper muscles, fascia,nerves, etc.
From the studies made of the spore-threads at different periods
during the eosin treatment, 3 a complete idea of the evolutions
through which the introduced spores pass has been obtained. A
certain small number of the spores germinate and grow into vegetative bacilli, which, after having reached this stage of development,
remain as such for a time, or, possibly, pass through a small seriesof divisions with the production of a few generations of bacilli.
The number of divisions is at best few, and the total number ofbacilli produced from the spores is relatively small. The vegetative bacilli degenerate within a few days, and the majority ofs N o g u c h i , " T h e N a t u r e of t h e A n t i t e t a n i c A c t i o n of E o s i n , " this n u m b e r ott h e JOURNAL.

Hideyo Noguchi.

295

the spores do not germinate at all, but remaining alive and quiescent, are enclosed in the scar tissue.
Doubtless, there is associated with the low degree of vegetationof the tetanus bacilli toxin production. This toxin, always smallin amount, is held chiefly in or near the locality in which it isformed by the surrounding wall of eosin through which it must,in the first place, pass slowly and with d!fficulty, and which, inthe next place, would tend to detoxicate it on its passage. The
effect, therefore, of the toxin would be exerted locally, if anywhere,
and if any cells capable of being excited to antibody-production
exist in the situation in which the toxin finds itself, they, presumably would be stimulated into activity. Should any of the toxin,
in a still active state, diffuse beyond the eosin barrier, it wouldfind itself in places from which it could readily pass into the bloodand into the internal organs, where it would be exposed to theusual conditions of antibody formation. I conceive that the mechanism of the antibody production in the particular case broughtout by these experiments is to be explained somewhat in the mannerof this supposition.
The questions immediately arise as to which cells in the local
tissues are concerned with the elaboration of the antitoxin, andwhether the antoxin as such is stored in these cells for immediate
liberation when called forth by the presence of the specific antigen.
_As regards the first question, no definitive answer can be given.
From the circumstances of the experiment, I am led to suppose theconnective tissue cells, possibly the endothelium of the lymphatics
as well, as the cells yielding the antibody. The extremely superficial character of the lesions must be kept in mind in attempting
to fix the parts the cells of which participate actively in the production of this local immunity. But I cannot bring forth proof
that the active cells are so strictly limited and so sharply circumscribed as I have indicated. It would, theoretically, seem to be lessdifficult to supply an answer to the second question. It does notseem to me highly likely that so diffusible a substance as antitoxinin the free state could remain in a state of high concentration
through many months in one part of the body at the same timethat it existed .in very low concentration elsewhere in the body.

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Imrro~nily to Tetanus in Inoculated Rats.

Oi1 the contrary, I should be more inclined to the view that a largepart of the general antitoxic inmmnity which the rats exhibited
may have been derived from the antibody absorbed from its local
site of production. The cells of the locally imnmne tissues have,I assume, undergone a physiological change which endures formany months, at least, enabling them to withstand the injurious
effects of tetanus bacilli and tetanus toxin either by means of rapid
liberation of antitoxic substances, or by an increased form of resistance to and destruction of poison (Giftfestigkeit) and bacilli withwhich the liberation of antagonistic antibodies is not necessarily
associated. Upon what remarkable changes in function or structure this power depends, we are in total ignorance, and any speculation must, therefore be wholly hypothetical.
The literature on immunity contains, as is well-known, otherexamples of local immunity. Doubtless in every case, at someperiod of antibody production, there exists a local immunity which
exceeds in degree that which the blood is able to display. This
fact must be acknowledged by all who believe antibodies not to
be formed chiefly in the blood itself. It will suffice merely to referto the observations of Pfeiffer and Marx 4 on the organs in which
the antibodies to the cholera bacillus are produced; to the experiments of Wassermann and TakakP of the fixation of tetanus toxin
by the brain and other organic tissues; to the studies of \Vassermann and Citron 6 on the local production by the pleural and peritoneal endothelium of antibodies (bacteriolytic, possibly othersalso) for the typhoid bacillus; to the interesting experiment ofvon Dungern 7 on the local production of precipitin for Maja serumin the anterior chamber of the eye; and, finally, to the observations of opthalmologists s upon the local immunity developed by
the conjunctiva to abrin inoculation, a phenomenon which R6mer
studied with great care and precision. Theoretically, there ismuch sirnilarity in all these observations, since they prove that antiZeit. f. Hygiene und Infektionskrank., 1898, xxvii, 272.
~Berl. klin. Woeh., 1898, xxxv, 5.
6 Zeit. f. Hygiene und Infektionskrank., 19o5, 1, 331.
~Die Antikorper, Jena, 19o3.
* Von Hippel, Archiv f. Ophthalmolog., 1883, xxix, 2I 3. Sattler, Klin. Monatsbl.
f. ,4ugenheilkr., 1883, xxi, 207. Neisser, Fortsch. d. Medicine, 1884, ii, 73. R6mer,Archly f. Opthalmologie, 19Ol, Iii, I72.

Hideyo Noguehi.

297

bodies are produced locally. The gradually increasing number offacts relating to this subject tend to exalt in importance cells which
hitherto have been regarded as indifferent in respect to antibody
production : namely, the cells of the connective tissues, lymph spaces,lymph vessels and serous cavities. The particular observations
which form the basis of this paper show that the antibodies to thetetanus poison can be produced in quantity by other cells than thoseof the central nervous system, for which, apparently, tetanus toxin
has an especial affinity.