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Journal of Experimental Medicine

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ETIOLOGY OF YELLOW FEVER.
XIV. DURATION OF THE PROTECTIVE EFFECT OF ANTI-ICTEROIDES

T~,o~cy~ S~Ru~ AFTER StmCtTTANEOtrS INOCt~LATION
n~To ~ L S .
BY HIDEYO NOGUCHI, M.D.

(From the h;boratories of The Rockefeller Institute for Medical Research.)(Received for publication, May 11, 1922.)

As has already been reported) the anti-icteroides immune serum iscapable of protecting susceptible animals against infection withLeptospira icteroides when administered simultaneously or during theearly period of the disease. Lyster, Pareja, Bailey, Vaughn, Vasconcelos, Casasus, Iglesias, Loyo, Le Blanc, Lynn and his associates,
Hernandez, Lara, Villamil, Kllgler, and Gann 2 have found thatthe mortality among human cases of yellow fever treated with theimmune serum on or before the 3rd day of disease is much lower
than that among untreated cases. Of 71 cases treated within thefirst 3 days of disease in Guatemala, Salvador, Honduras, Vera Cruz
Merida, and northern Peru only 5 died (7 per cent mortality); theresults of serum treatment were less favorable in the Tuxpam (Mexico)
epidemic, where the mortality among the treated was 25 per cent
(9 deaths among 36 cases), but here the mortality among untreatedcases was correspondingly higher, being 68.6 per cent (59 deathsa Noguchi, H., Serum treatment of animals infected with Leptospira icteroides
J. Exp. Med., 1920, x.x.xl, 159; Chemotherapy versus serotherapy in experimental
infection with Leptospira icteroides, 1920, xxxii, 381. Noguchi, H., and Kligler,I.J., Immunologicalstudies with a strain of leptospira isolated from a case ofyellow fever in Merida, Yucatan, J. Exp. Med., 1920, xxxii, 627; Immunology of
the Peruvian strains of Leptospira icteroides, 1921; xxxiii, 253.
t Noguehi, H., Prophylaxis and serum therapy of yellow fever, J. Am. Meal.
Assn., 1921, lxxvii, 181. See also the report of The Rockefeller Foundation forthe year 1921.
357

358

ETIOLOGY O1~ YELLOW ~'EVER.

XlVamong 86 cases). Of the total 187 cases treated to date, 107 received
the serum on or before the 3rd day, and of these, only 14 died (mortality 13 per cent); on the other hand, there have been 41 deaths (51per cent mortality) among 80 cases treated on or after the 4th day,and 225 deaths (56.6 per cent mortality) among 397 untreated cases
occurring in the same localities during the same epidemic periods.
The serum has apparently undoubted therapeutic value in humancases of yellow fever as well as in cases of experimental infection with
Leptospira ict~roi,des in animals. Vaccination by means of killedcultures of Leptosplra icteroides (injected in two subcutaneous injections of 2 cc. each, 4 to 6 days apart) has been shown to confer complete protection within 10 to 15 days of the last inoculation.2,a During the period required for development of active immunity, however,anti-icteroides serum might be utilized for the immediate protectionof non-lmmune individuals who find themselves in an epidemic orendemic focus of yellow fever, or it might be substituted for vaccination in the case of persons who intend only to pass through an infecteddistrict. It is well known that an immune serum or antitoxin, whenintroduced into the system of a non-immune individual, will protect
against infection for a period which varies from a few to many days
according to the number of units of immune bodies initially introduced
and the rate of elimination of such bodies from the inoculated individual. The passive immunity thus conferred is necessarily of
short duration, yet the employment of anti-icteroides serum for the
temporary protection of non-immune individuals might be of considerable practical value.
In the experiments to be reported here guinea pigs were used todetermine the duration of the protective effect of an injection of antiicteroides immune serum.
EXPERIMENTAL.

Six different doses of immune serum (0.00001, 0.0001, 0.001, 0.01,0.1, and 1 cc.) were subcutaneously inoculated into six sets (two each)of guinea pigs of about 500 gm. body weight. The tests for the per*Noguchi, H., and Pareja, W., Prophylactic inoculation against yellow fever,J. Am. Med. Assn., 1921,lxxvi, 96.

HIDEY0 NOGUCHI

359sistence of passive immunity were made 1, 2, 3, 4, 5, 7, 10, and 15
days after the injection of the immune serum. In order to infect all
the guinea pigs, including six normal control animals, with the samematerial at the same time, the injections of the immune serum werebegun 15 days before the time selected for the test inoculation of avirulent strain of Leptospira icteroides. The infective material used
was an emulsion of the liver and kidneys of a guinea pig fatally infected with a strain of Leptospira icteroides isolated in Morropon byNoguchi and Kligler, 4 which killed the control guinea pigs within 9
to 10 days in quantities of 0.001, 0.01, and 0.1 cc. 0.1 cc. of theemulsion, representing about 100 minimum lethal doses, was used inthis experiment and was given subcutaneously. The results arerecorded in Table I.
Analysis of the table shows that the minimum quantity of antiicteroides serum required for protection, as revealed by the results
obtained when the serum was given an hour previous to the inoculation of the infecting material, lay between 0.00001 and 0.0001 cc.
Judged from the survival of three guinea pigs which received serum
24, 48, and 72 hours previously the protective titer of the serum
must be close to 0.0001 cc. There was no diminution in the protective
effect within 48 hours, but after 3, 4, and 5 days 0.0001 cc. no longer
gave complete protection, while 0.001 cc. protected in every instance.
7 days after the time of injection of the serum 0.001 cc. failed to protect, but 0.01 cc. was still effective. After the lapse of 10 days 0.1 cc.
was required to prevent infection, and after 15 days only those animals
which had received 1 cc. of the immune serum withstood infection.
The rate of elimination of the immune substance in the body of theguinea pig after the subcutaneous introduction of the anti-icteroides
serum does not proceed uniformly in the successive days following
injection, but follows a characteristic course which may be roughly
estimated in the manner shown in Text-fig. 1, the unit being 0.0001
cc., which neutralized at least 100 minimum lethal doses in thepresent series.
The rate of disappearance of the immune substance is very slow atfirst, but becomes rapid after about 10 days. This phenomenon is4Noguchi, H., and Kligler, I. J., Experimental studies on yellow fever innorthern Peru, J. Exp. Med., 1921,xxxiii, 239.

ETIOLOGY OF YELLOW FEVEI~.

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not characteristic of anti-icteroides serum alone, because various
immune serums appear to undergo a similar reduction in strength
when introduced into different species of animals. Knott, forexample,
found that tetanus antitoxin (horse serum), given to a foreign species(guinea pigs or rabbits), is reduced rapidly and that only about ~-~Units

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of the quantity originally present in the blood can be demonstrated
after 12 to 14 days. 5 Tizzoni s obtained similar results. Nocard 7
estimated that the longest period that the tetanus antitoxin remains
Knorr, A., Das Tetanusgift und seine Beziehungen rum thierischen Organismus. Eine experimentelle Studie fiber Krankheit und Heilung, Mt~nch. rned.
Woch., 1898, xlv, 362; Die Entstehung des Tetanusanfitoxins im ThierkSrper undseine Beziehung rum Tetanusgift, Fortschr. Med., 1897, xv, 657.
6Tizzoni, G., Sull' efficaciadell' anfitossina nel trattamento prevenfivo contro
il tetano dopo awenuto l'infezione, Gazz. osp., 1897, xviii, 1215.
7 Nocard, E., Sur la s6roth6rapie du t6tanos; essais de traitement prevenfif,Bull. Acad. reed., 1895, xxxiv, 407.

Hm~.~O Noovc~x363

in the body is about 4 to 6 weeks. Pfeiffer and Friedberge# foundthat when an anticholera serum derived from the goat is injected into
the rabbit it gives rise to an anti-immune substance by which it isneutralized in a comparatively short time. Ransom and Kitashima °showed that homologous tetanus antitoxin, when injected into anormal horse, may remain as long as 80 days; that is, the duration of
the passive immunity is almost as long as that of active immunity.
On the other hand, J6rgensen and Madsen found that not all homologous immune serums remain for a long period in the body ofthe injected animal, but that the length of time varies with different
animal species.10 Kraus and his coworkers failed to demonstrate
any evidence for the formation of an anti-immune substance in theblood or organs of animals which had received an injection of aheterologous diphtheria antitoxin, n Perhaps the sudden disappearanceof the immune substance after about 10 days in the present seriesof experiments may be intimately connected with the precipitin formation for the heterologous anti-icteroides horse serum. Moreover,
the titer of the immune serum is certain to suffer reduction when
kept at a temperature of 39°C., even in vitro, and may be expected
to undergo similar reduction in the blood of a foreign species.
S~ARY.

Analysis of the records of instances in which non-immune persons
contracted yellow fever notwithstanding vaccination shows that theonset of disease occurs soon after vaccination, the longest period being
13 days. Since the average incubation period in yellow fever is 6 days,
it seems that infection must have taken place in some instances
during the period while protection was developing. These instancess Pfeiffer, R., and Friedberger, E., Ueber den Verbleib der bacteriolytischen
lmmunk~rper im tierischen Organismus nach der passiven Immunisierung, Cen~r.
Bakt., 1re AbL, Orig., 1904, xxxvii, 131.
9 Ransom, F., The conditions which influence the duration of passive immunity,
J. Path. and Bact., 1900, vi, 180.
10 J/irgensen, A., and Madsen, T., The fate of typhoid and cholera agglutinins
during active and passive immunisation, Festskrift red Indvielsen af Statens
Serum-Institut, 1902, Copenhagen, Paper 6.
11Kraus, R., and Joachim, J., Zur Frage der passiven Immunisierung, Wign. klln.
Woch., 1903,~xvi,1389.

364

ETIOLOGY O F Y E L L O W F E V E R .

XIV

led to a study of the possibility of immediate protection by means of
the anti-icteroides serum. It had already been shown that the immune serum protects at once against experimental Leptospira icteroides
infection, but it remained to determine how long the protection wouldlast.
Guinea pigs were given different quantities of the immune serumand subsequently injected, at various intervals, with a virulent strainof Leptospira icteroides. Complete protection enduring 5 days wasobtained with as minute a quantity of serum as 0.002 cc. per 1,000gin. of body weight. After 5 days, however, the immune substance
rapidly diminished~ and to keep the animal protected for as long as
10 days it was necessary to give 100 times as much, or 0.2 cc. For
a man weighing 80 kilos, 0.16 cc. (0.002 X 80) would theoretically besufficient to protect for at least 5 days, 1.6 cc. for 7 days, and 16 cc.
for 10 days. This temporary protection may be a valuable antecedent
to that furnished by vaccination, since the final effect of the lattercannot be expected until at least 9 to 10 days have passed.