野口英世著

Journal of Experimental Medicine

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A COMPARATIVESTUDY OF EXPERIMENTAL
PROPHYLACTIC INOCULATIONAGAINST LEPTOSPIRAICTEROHz~EMORRHAGI~E.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The RockefellerInstitute for Medical Research.)(Received for publication, August 16, 1918.)
Ido, Hoki, Ito, and Wani ~first demonstrated that when guinea pigs are treatedwith three successive injections of killed cultures of Leptospira icteroh~emorrhagi6e
at 5 day intervals they become refractory to a subsequent infection, undertaken
10 days later, with a virulent culture or with liver emulsions of infected guinea
pigs. After about 17 to 19 clays the serum of these animals contained a small
amount of immune bodies when tested by the method of Pfeiffer. From thisexperiment the conclusion was drawn that the degree of immunity induced inthese animals is comparable with that which develops in the serum of patients on
the 8th day of disease, in the stage when the infective organisms have alreadydisappeared from the peripheral blood, as shown by negative results of animal
inoculations with the blood. The immunity is incomplete, but sufficiently strongt o protect the animals from the infection. These writers found also that the serumof six persons inoculated with three successive doses of highly concentrated vaccine(carbolized emulsion of the pure culture) for the purpose of prophylaxis contained
a demonstrable amount of immune bodies when tested by Pfeiffer's method 10
days after the last inoculation, but when given to unvaccinated guineapigs theserum failed to protect the animals against a simultaneous infection. There wasconsiderable difference, however, between the course of the disease in the animals
receiving the serum from vaccinated persons and those receiving normal humanserum, the former outllving the controls by as many as 10 days, the number ofspirochetes being fewer, and the icterohemorrhagic lesions less marked.
Ito and Matsuzaki 2 recommended for the purpose of protective vaccination theuse of the blood gelatin culture of Leptospira icterohcemorrhagice. The culture waskilled by heating it to 60°C. for 30 minutes on 3 successive days. The killed culture was toxic when given intravenously, and one of the samples killed guinea pigs,
in a dose of 0.5 cc. or more per 100 gm. of body weight, within a few minutes. A
single subcutaneous injection of 0.05 cc. of another preparation of the killed cul1 Ido, Y., Hoki, R., Ito, H., and Wani, H., The prophylaxis of Well's disease(spiroch~etosis icteroh~emorrhagica), J. Exp. Med., 1916, xxiv, 471.
2 Ito, T., and Matsuzaki, H., Prophylactic vaccination, serotherapy, and chemotherapy in Well's disease (in Japanese), J. ChibaMed. College,1916, Nos. 83 and 84.
561

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LEPTOSPIRA ICTEROH~;M 0RRttAGI.'E

ture, the minimum lethal dose of which was 1.5 cc. per I00 gin. of body weight, wasfound to be sufficient to confer upon the animal a complete,immunity against asubsequent infection after 2 Weeks, although there was no protection at the end of
the 1st week. Ito and Matsuzaki also prepared a pulverized vaccine from a.desiccated blood gelatin culture of the icterohcemorrhagi~eand found that it not
only retained its immunizing quality but possessed the advantage over the liquid
vaccine of being nearly ten times more concentrated. For protective vaccination
they used a saline emulsion of the dried culture. They calculated the quantity
necessary for a man of 50 kilos body weight to be 25 to 30 cc. of the liquid and 2.5
to 3 gin. of the dried material. The serum from the vaccinated animals contained
the specific immune bodies demonstrable by Pfeiffer's procedure and also in vitro(Sabritschewsky's phenomenon).
F r o m the experiments of Ido, Hoki, and others on the one hand, andthose of I t o and M a t s u z a k i on the other, it is evident t h a t the guinea
pig, which is perhaps the animal most susceptible to infection b y
Leptospira icterohcemorrhag#e, can be rendered actively immune b y asingle injection or b y multiple injections of devitalized cultures. T h equestion arises as to whether the i m m u n i t y thus induced with onestrain will p r o t e c t the animal equally well against the other strains.
T h e present work was u n d e r t a k e n to determine the relation of one
strain to another b y means of cross-immunity reactions among thestrains isolated in Japan, America, and Europe.

Method.
For the purpose of protective inoculation guinea pigs weighinga b o u t 400 gin. were selected. The emulsions of Leptospira icterohcemorrhagice were prepared from pure cultures of different strains
grown on the rabbit serum Ringer medium (semisolid below andliquid above 3) at 26°C. for from 3 to 6 weeks. T h e liquid portion of
the culture, showing 20 to 30 organisms per field (t~ oil immersion
and Leitz ocular 3), was gathered from a n u m b e r of tubes and, after
immobilization of the organisms b y means of 0.4 per cent phenol(determined b y the dark-field microscope), was centrifuged for 2 hours
a t the rate of 4,000 revolutions per minute; the deposit was carefully
separated from the s u p e r n a t a n t fluid and resuspended in one-fiftha Noguchi, H., Further study on the cultural conditionsof Leptospira (Spiro-ch6eta) icteroh6emorrhagice,J. Exp. Med., 1918, xxvii, 593.

HIDEYO NOGUCHI563of the original volume. The emulsion thus concentrated contained agreat number of the organisms and was preserved under a layer oftoluene for 1 week in the refrigerator (6°C.) before use. The emulsion was then tested b y culture in order to be certain that there wereno live organisms left.
Emulsions were prepared from two American strains isolated fromwild rats caught in the vicinity of New York, one European, and one
Japanese strain,* and the inoculations were made subcutaneously.
In the first group of animals 0.5 cc., in the second 0.05 cc., and in thethird 0.005 cc. of the emulsion was given three times at intervals of 5days. Except in a few instances the animals showed no rise in temperature after the inoculation. As a whole they stood the inoculations
well, though a number were lost through secondary infection. Some 'lost weight during the vaccination period.
Tests for active immunity against the homologous and heterologous
strains were made at the end of 2, 4, and 8 weeks after the last inoculation. The quantities used for the injection intraperitoneally into
the vaccinated and control (unvaccinated) animals were those containing at least several minimum lethal doses of each strain. For
example, in the case of American Strain 1,0.0005 cc. was used, American Strain 2,0.005 cc., American Strain 3,0.05 cc., Japanese, 0.00005
cc., and European, 0.005 cc. of 3 week cultures on the rabbit serum
Ringer medium at 26°C. Two control animals accompanied eachseries of experiments, and they always died of the typical infection
within 5 to 7 days.
Notwithstanding the care which they received, some of the vaccinated guinea pigs died of secondary infection, or in a few instances ofmarasmus, before the experiments were completed.
RESULTS.

The results of the tests are briefly summarized in Tables I to IV.
Table I shows that the guinea pigs receiving three successive injections of 0.05 and 0.5 cc. of the concentrated vaccine of American
Strain 1 were, with two exceptions, protected against a subsequent
4Noguchi, H.,Spirochcetaicterohcemorrhagi¢inAmerican wild rats and its relation
to the Japanese and European strains, J. Exp. Med., 1917, xxv, 755.

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infection undertaken 2,4,and 8 weeks after the last inoculation. One
exception occurred in the case of American Strain 2 and one in the caseof the European strain, both animals succumbing to the infection 1 to2 days later than their respective controls. On the other hand, three
injections of 0.005 cc. of the same vaccine protected the animals against
the same strain as well as against American Strain 3, but failed to produce complete immunity against the other three heterologous strains.
Table II gives the results obtained with the guinea pigs vaccinatedwith American Strain 2. The protection was complete in the group
receiving 0.05 and 0.5 cc., except when tested against the Japanesestrain and American Strain 1, in which instances death was delayed
but not prevented. There was no resistance against any strain when
the dose of the vaccine was 0.005 cc.
In the series reported in Table III, the highest degree of immunity
is seen to have been developed against the Japanese strain, which was
the one used as the vaccine in this series. It was the only strain which
failed to kill guinea pigs that received injections of the smallest
amount of the vaccine (0.005 cc.). Protection was weakest against
American Strain 2 and the European strain. Three injections of 0.5
cc. in succession, however, conferred complete immunity against all
the strains tested.
A similar relation exists between the homologous and heterologous
strains of the next series (Table IV), the protection being most markedagainst the same organism. All the guinea pigs vaccinated with 0.5
cc. of any one strain resisted infection with that and all other strainsused in the present study.
While these results do not furnish basis enough to permit division of
the various strains into groups or definite types, yet the general indication is that there is a closer affinity among some strains than others.
For example, American Strains 1 and 3 seem to be closely allied,
American Strain 2 being nearer to the European strain. The Japanesestrain stands between the two groups and much nearer that formed byAmerican Strains 1 and 3.
With respect to the duration of the immunity thus established, it
was found that at the end of 8 weeks there was not an appreciable
diminution of the immunity, although of the animals receiving smaller
amounts of the vaccine a slightly larger number survived when tested
at the end of 4 than at the end of 2 or 8 weeks.

HIDE'gO NOGUCHISUMMARY

AND569CONCLUSIONS.

Guinea pigs were inoculated with suspensions of Leptospira icterohcemorrhagice obtained from pure cultures of several different strains,in order to determine whether or not an active immunity against a subsequent infection with virulent organisms would develop in the vaccinated animals. The experiments were so arranged as to make possible a determination of the existence of immunity against homologous
strains as well as against the strains not employed as vaccine, and abrief quantitive estimation of the degree and duration of the immunity
in relation to the quantities of the vaccines inoculated. Following thegeneral rule of prophylactic inoculations with various pathogenic organisms, the inoculations were repeated subcutaneously on three consecutive occasions at intervals of 5 days. With respect to the amountsof vaccine, the experiments were divided into three groups foreacti
vaccine, one group receiving three doses of 0.5 cc., the second three of
0.05 cc., and the third three of 0.005 cc. Four different strains wereemployed as vaccines, American Strain 1, American Strain 2, and one
each of the Japanese and the European strains.
The determination of the development, degree, and duration of theimmunity was made by inoculating intraperitoneally several minimum
lethal doses of each of the five following strains: American Strains 1,2, and 3, the Japanese, and the European strains. The virulence of thedifferent strains varied considerably, the strongest being the Japanesestrain, which killed the guinea pig in a dose of 0.00001 cc., and theweakest American Strain 3, the minimum lethal dose of which was aslarge as 0.01 cc.
The vaccinated guinea pigs were tested for immunity at the end of
2, 4, and 8 weeks after the last inoculation.
The results obtained show that three successive inoculations of0.5 cc. of the emulsions of killed cultures of Leptospira icterohcemorrhagice into guinea pigs rendered them completely resistant to a subsequent infection with the virulent cultures of both homologous andheterologous strains. With 0.05 cc. the protection was not so general,
the animals succumbing to an experimental infection with some heterologous strains while resisting the homologous and other heterologousstrains. Ttie animals which were vaccinated with 0.005 cc. survived

570

LEPTOSPIRAICTEROHIEMORRHAGI2E

the infection experiments with the homologous strains in the case ofAmerican Strain 1 and the Japanese strain, but they were not protectedagainst any other strains. The vaccines of other strains were unable
to immunize the guinea pigs so highly even against their homologous
strains, when the amount of each inoculation was only 0.005 cc., but
0.05 cc. conferred complete protection against the same strains. It
may be concluded, therefore, that.when a sufficient quantity of killedcultures of Leptospira icterohcemorrhagi¢e is given, the guinea pigs
will become immune to all strains of the same organism, but that
smaller quantities may protect them against homologous but notagainst heterologous strains. A close analysis reveals the existence ofgroup or t3~pe affinities among different strains which can be brought 'out by immunizing the animals with smaller quantities of killed cultures. In the present series of experiments American Strains 1 and 3
form one group, American Strain 2 and the European strain another,and the Japanese strain a third, which is also closely allied to the firstgroup.
In order to insure universal immunity it is wise to employ as manygroup or type cultures as possible in the preparation of vaccines, apolyvalent vaccine being recommended. I t is not improbable thatthe strain recently encountered in Lorient, France, 5-8 is an unusually deviated type of Leptospira icteroheemorrhagice, and that if
successfully cultivated and used as vaccine in sufficient amount it
might protect the animals against other strains of the same organism.
The active immunity induced in the vaccinated guinea pigs wasfound to persist for at least 8 weeks after the last inoculation. It will
no doubt last for a much longer period.
5Manine, Cristau, and Plazy, Compt. rend. Soc. biol., 1917, lxxx, 531.
6Manine and Cristau, Bull. el m~m. Soc. m~d. hop. _Paris, 1917, xli, series 3, 977,1045.
7 Pettit, A., Compt. rend. Soc. biol., 1917, lxxx, 774.
8Pettit, A., Compt. rend. Soc. biol., 1918, lxxxi, 48.