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Journal of Experimental Medicine

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I M M U N I T Y STUDIES OF ROCKY MOUNTAIN SPOTTEDFEVER.
I I . PROPB'YLACTIC INOCULATION IN ANIMALS.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, June 26, 1923.)

Of the many urgent and interesting problems regarding Rocky
Mountain spotted fever, that of personal prophylaxis seems to be themost important. The experiments reported here were carried out
in an attempt to devise a practical method by which a non-immune
population might be rendered immune to the bites of infected ticks.
Ricketts and Gomez1 early recognized the possibility of developing
a satisfactory method of preventive inoculation, and carried out a few
experiments in this connection but no adequate quantitative experimental analysis of the factors concerned in the production of artificial
immunity. Since we have no pure culture of the etiological agent andcannot recognize it microscopically, we must rely solely upon animal
inoculations for quantitative as well as qualitative estimation of thepresence of the spotted fever organism. Since the number of causative
organisms must bear a definite relationship to the amount of antibodies or the degree of immunity which is produced, a knowledge ofthe number and virulence of the organisms used is a prerequisite to anunderstanding of the phenomena of immunization. For example,
1 cc. of the citrated blood of guinea pigs with all the symptoms of
spotted fever may contain only 1 or as many as 1,000 minimum lethal
doses, but this fact can be ascertained only by careful titration of
the material by animal inoculations, and unless the number ofminimum lethal doses is determined in each instance, the immunization
experiments carried out become meaningless or even misleading.
Moreover, since the virus dies out in citrated or defibrillated blood
1 Ricketts, H. T., and Gomez, L., Studies on immunity in Rocky Mountainspotted fever, J. Infect. Dis., 1908, v, 221.
6O5

606

ROCKY MOUNTAIN" SPOTTED F E V E R .

IIor serum, even at 4°C., it must be titrated simultaneously with thevaccination or subsequent immunity tests in which it is being used.
The selection of experimental animals is another important factor,
and since our aim is to develop methods of protection for the humanbeing, we must choose an animal in which the morbidity and mortality
with respect to spotted fever infection approach as closely as possible
those of man. The guinea pig excels other animals in this respect.
The rabbit is far less susceptible than the guinea pig, and monkeys(Macacusrhesus) are extremely susceptible, those of a series of eighteen
having shown 100 per cent morbidity and mortality. The age and sizeof the animal are very important. Full grown males, weighing 500 to
600 gm., are to be recommended; first, because of their comparatively
greater resistance to banal laboratory infections and their undiminishedand remarkably constant susceptibility to the spotted fever infection,and second, because the characteristic genital lesions are best observedin male animals. The consideration of such factors as just outlined isabsolutely essential for formulating a practicable method of prophylactic vaccination.
The ordinary immunological procedures, known to be effective inother diseases, have been subjected to experimental determination of
their possible application in the case of spotted fever: (1) the effectof injection of non-infective quantities of live spotted fever virus;(2) the effect of injection of killed virus; (3) the effect of injection ofdead virus; (4) the effect of injection of freshly prepared neutral orsuperneutral mixtures of the live virus with immune serum; (5) the
effect of heating and of age upon the immunizing properties of neutral
mixtures of the virus with immune serum.
The two strains of spotted fever virus used in the present series ofexperiments were obtained through the kind cooperation of Dr. R. R.
Parker, ~ud we continue to use his designation of them as "Cooper"and "Barlow." The guinea pigs were almost always adult males
weighing 500 to 600 gin. The virus was used in the form of citrated
blood (equal parts of blood and citrate solution), usually drawn fromthe heart of the infected guinea pig on the 4th to 6th day of fever,preferab|y when the scrotal lesions became noticeable. Blood drawn
on the 2rid or 3rd day of fever contains, in my experience, far less ofthe virus. Every specimen of blood was tested for bacterial contami-

607

HIDEYO NOGUCHI

nation on ordinary broth and slant agar and was titrated simultaneouslywith each experiment to determine the n u m b e r of killing doses whichit contained. All the injections of virus, or mixtures containing virus,
were m a d e intraperitoneally. T h e temperatures of the animals weretaken every morning during the period of observation; t h a t is, untild e a t h or recovery, or, in case no infection developed, for a t least 2
weeks. T h e immune serums used in this work were chiefly fromTABLE I.

Effect of Non-Infectious Quantities of Virus.
Date o[inoculation.

Material.

Amount.

Reset.

Immunity test.

No reaction.

June 13, 1922 (33 days)
Cooper strain 1 cc. (1,001~.L.D.); typical infectio]
but recovered.
Similarly tested; typical in
fection; death in 11 days
Similarly tested; typical in
fection; death in 11 days
Feb. 17, 1922 (23 days)
Barlow virus 0.5 cc. (~,J.r..D.); typical infection
death in 10 days.
Similarly tested; typical infeetion; death in 19 days

c6.

19ZZ

May 10. Citrated guinea pig 0.001
blood, Cooperstrain (1 ~.L.D.
= 0.01 cc.).

0.00010.00001
Jan. 25. Citrated guinea pig 0.1
blood, Barlow
strain (1 ~.L.D.
= 1 co.).

0.01

rabbits which had passed through a typical but non-fatal infection
and had been bled 2 to 3 weeks after the temperature returned tonormal3 Scrotal lesions were frequently observed in the infected
rabbits. In some instances the immune serums came from rabbits
which had received a second dose of virus (4 cc. citrated blood of theguinea pig) during convalescence; these serums are designated as"reinforced," and their titers are somewhat higher than those of theconvalescent serums. AI] the immune serums were k e p t in the
refrigerator at 4°C. except at time of use.
Noguchi, I-I., Immunity studies of Rocky Mountain spotted fever. I. Usefulness of immune serum in suppressing an impending infection, J. Exp. Med.,
1923, xxxvii, 383.

ROCKY608~OUNTAINSPOTTED FEVER.

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ROCKY MOUNTAIN SPOTTED F E V E R .

II

Effect of Non-Infective Quantities of Live Spotted Fever Virus.
That guinea pigs which survive a non-fatal but typical spotted
fever infection resist subsequent infection has long been known, butwhether or not animals receiving too small a quantity of the virus
to become infected would acquire increased resistance remained to
be determined. Ricketts and Gomez published some observations
on this point, 1 stating that, although they had made no systematic
attempt to determine the vaccinating properties of minute doses ofvirus, they had reason to believe that prophylactic vaccination could
not be based on this principle. The present experiment shows (TableI) that no appreciable immunity develops under these conditions.
Three guinea pigs received non-infective doses of one strain and twoof another, and all succumbed to subsequent inoculation 33 and 23days later, respectively.

Effect of Injection of Virus Killed Either by Chemicals or by Heating.
Chloroform, ether, and xylene were allowed to act upon the virusby shaking with it for 5 minutes. After the mixtures had been in the
refrigerator (4°C.) for 20 hours, the clear portion in each case wasused for inoculation. Guinea pigs were given the sterilized virus indoses of 1, 0.3, and 0.1 cc., and the animals were tested for immunity
28 days later by inoculation of a single minimum lethal dose of Barlow
strain (0.1 cc.). There was no evidence of any increased resistance tothe infection.
Another portion of the same virus was heated to 56°C. for 10 minutes
in a water bath. Three guinea pigs were then inoculated with 0.75,
0.3, and 0.1 cc., respectively. No infection followed, and 28 days
later each animal was given one minimum lethal dose of the virus(Barlow strain, 0.1 cc.). All had severe and typical infections, one
dying in 14 days (Table II).
The failure of the animals to derive any immunity from the introduction of the killed virus was apparently due to modification of theimmunizing substances by the chemical or physical agents, since thesame quantities of virus (100, 30, and 10 ~r.L.D.) when mixed withimmune rabbit serum, proved ample, as will be shown later, to renderanimals immune to the inoculation of the same test material.

Hm~Yo NoGvcm

611

Effect of Injection of Dead Virus.
Although the foregoing experiment had shown that the virus could
not be killed without impairment of its immunizing properties, thequestion remained whether it might not retain its power to confer
immunity if allowed to die out under such unfavorable conditions as,
for example, low temperature for a long period. Hence several
animals, which had been inoculated with specimens of virus kept in
TABLE III.

Effect of Injection of Dead Virus.
aStrain.
perkofAmount,

day

co.

69

2

Same

69

CooperGuinea pig 19
CooperGuinea pig 30

57

4 (of 1:6 solution in citrate).
2

CooperGuinea pig 31
CooperGuinea pig 47
Barlow
Guinea pig 24

28
2I
38

Result of immunity test,
40 days afterinoculation,
against Barlow virus
1 cc. ( 1 0 0 M. L.

CooperGuinea pig 11

31

Result of inoculation.

I).).

No reaction; diedof secondary infection in 9 days.
No reaction.
Typical infection;
died in 16 days.
Typical infection;
died in 12 days.
Typical infection;
killed for material.
No reaction.

Typical infection; finaUy recovered.
Typical infection;
died in 15 days.
Typical infection;
died in 12 days.

the refrigerator (4°C.) for periods of 28 to 69 days and had had noinfection, were tested 40 days later for immunity by the injection of
100 ~t.L.D. of virus (Barlow strain, 1 cc.). The animals all developed
typical and, in most instances, fatal infections (Table III. Compare
also Table IX, under the heading "Controls (heated virus alone).").

612

ROCKY

I ~ O U N T A I N SPOTTED F E V E R .

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614

ROCKY MOUNTAIN SPOTTED F E V E R .

II

Effect of Injection of Freshly Prepared Neutral or Superneutral Mixturesof Live Virus with Immune Serum.
I t had been noticed early in these studies of immunity in spotted
fever that guinea pigs which had received suitable mixtures of virus
and immune serum remained well throughout several weeks of observation. The mixtures were innocuous, irrespective of the amount ofvirus introduced, provided the amount of immune serum were correspondingly adjusted. For example, 1 cc. of virus containing 100 or
1,000 M.L.D. might be injected without causing any infection whenmixed with 0.1 cc. or more of immune serum, and animals so inoculated
were found to be completely refractory to subsequent inoculation withas many as 100 or 1,000 ~t.L.I). The degree of immunity acquired was
altogether comparable with that of animals which had received asubneutral mixture and had passed through a mild course of fever or amoderately severe infection with scrotal lesions. The immunity
produced by subneutral mixtures is apparently the same as that following convalescence in animals which survive a typical severe infection
caused by virus alone.
Table IV shows the neutralizing power of the serum of eight convalescent rabbits and of four hyperimmunized immune rabbits (i.e.
which had received a second injection of virus during convalescence)
in 0.1 and 0.01 cc. against 100 M.L.D. (Cooper strain, 1 cc.) and thedegree of immunity developed in the guinea pigs. 0.1 cc. of the convalescent serum was an almost, b u t not completely, neutralizing dose,
while the same quantity of the reinforced immune serums was sufficientto neutralize all infectivity. The guinea pigs were tested for immunity
on several different occasions and with virus of various degrees ofvirulence. Of the eight animals tested 33 days later with 1,000~r.L.i)., none became infected, and no change in temperature wasobserved. Two tested 3 months afterwards against 1,000 M.L.D.
resisted infection. One animal was tested after 6 months with 100~r.L.I). and proved resistant. Several others were tested after longer
periods--8, 9, and 10 months--against 1, 5, and 30 ~t.L.D. of virus andlikewise proved immune.
Superneutral Mixtures.--In the case of human vaccination theneutral mixtures would leave too narrow a margin between theHmEYO NOGUCHI

615

neutral and the subneutral, and no margin can be too great to insure
safety for the persons vaccinated. Since, however, a great excess of
the immune serum in a mixture might reduce or even nullify the
immunizing properties of the virus, it was necessary to determine thispoint. Hence a series of experiments was carried out in which a
TABLE V.
Effect of I n j e c t i o n of S u p e r n e u t r a l M i x t u r e s of V i r u s a n d I m m u n e S e r u m .

M a y 25, 1922. Virus: citrated guinea pig blood (mixed Cooper a n d Barlow
strains), 1 ce. (100 ~.L.D.). I m m u n e serum" rabbit.
I m m u n i t y test.
Immune$erunlNo.

Anlol] at.

Vaccination
reaction.

Intervalafter
vaccination.

Degree of p r o t e c t i o n .

¢C.

11
23
25

1

Mild fever; recovery.
No reaction.

7 mos.

Complete against 1 M.L.D,

18 days.

Died of intercurrent infection; no lesionof spotted fever after injection of 1,00q

1 too°

Complete against 100 M.T.D.
""
1,000 "
"
"
100 "
"
"
1,000 """
1,000 """
1,000 "

~ . L . D . V~rus.

186

4 mos.
307
314
316
326
327
329
330

1 too.
~gcc

4 mos.
4 "4aReserved for later test.
cg

ct

I

4 mos. i Complete against 1,000 u.r..D.

7 "
332
349
350

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7½
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considerable excess of the serum was assured b y using 1 cc. against
100 M.L.D. of virus. Fourteen different samples of immune serum,
twelve of which been used also in the previous series of experiments,
were used (Table V), all but one of which (No. !1) completely neutralized the virus. The animals were tested for immunity after
1, 4, 7, and 7½ months, respectively, and found to be completely

616

I~OCKY M O U N T A I N

SPOTTED

I~EVER.

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618

ROCKY

MOUNTAINSPOTTED

FEVER.

II

resistant to infection with virus containing in some instances as m a n y
as 1,000 m i n i m u m lethal doses.
The results showed t h a t the i m m u n e serum m a y be added to thevirus in great excess w i t h o u t u n f a v o r a b l y influencing the developmentof immunity.
TABLE VII.
Durationo f _Passive I m m u n i t y .

June 24, 1922.
Immuneserum(rabbit) No.

__
23

Result.

Immunitytest.

N o reacfion,.

Tested 12 days later against Cooper virus
1 cc.;* typical infection; died in 9 days.
Tested 12 days later against Cooper virus,
1 cc.;* mild infection; recovery.
Tested 2 months later against Coopervirus, 1 cc.
(1,000 M.L.D.); no infection.
Tested 2 months later against Cooper virus,
1 cc. (1,000 ~.L9.); typical severe infection;
died in 11 days.

17_
1

~c

25

~c

Titration of Cooper strain virus used for immunity tests. September 25, 1922.
Guineapigcitrateblood.

Resultofinoculation.

co.

0.5
0.1
0.01
0. 001
0. 0001

Severe infection with scrotal lesions; killed forvirus in 10 days.
Severe infection with scrotal lesions; killed forvirus in 10 days.
Severe infection; died in 13 days.
""" " 13 "No infection.

* Control died in 14 days.
T o confirm a n d supplement the above experiments, another seriesof animals was inoculated with mixtures containing the same a m o u n t ofvirus with v a r y i n g aznounts df i m m u n e serum. T w o i m m u n e serums
were separately used, and for each dose two animals were inoculated.
2, 1, 0.5, 0.25, and 0.1 cc. of serum, respectively, were mixed with 1
cc. of virus containing 100 m i n i m u m lethal doses or a b o u t 1,000 minim u m infecting doses. N o reaction followed the inoculation in theHmEYO NOGUCHI

619

animals which received the m i x t u r e containing 0.5 cc. or m o r e of serum,b u t animals which received 0.25 cc. or less all h a d so.me degree offebrile reaction, although no scrotal lesions were noted. I r r e s p e c t i v eof w h e t h e r the q u a n t i t y of serum was subneutral, neutral, or superneutral, all the animals p r o v e d to be equally resistant to severals u b s e q u e n t a t t e m p t s to infect t h e m a f t e r intervals of 3 m o n t h s(with 1,000 ~.L.D. of virus), 5, 5½, 6, and 7 m o n t h s (with 1 to 5 ~.L.D.
TABLE

VIII.

Protective Power of the Blood of Vaccinated Guinea Pig.

Date of vaccination, May 25, 1922.
Material: virus (Cooper and Barlow strains mixed), 1 cc. + immune rabbit
serum, 1 cc.
This animal belongs to the series reported in Table V.
Intervalafter
vaccination.

Test of blood for protective power ofcitrate plasma.*Result of inoculation.

1 cc. + virus, 0.1 cc. (10 ~r.L.D. or
100 M.I.D.)
0.2 CC. + same virus, 0.1 cc.

Definite febrile reaction; recovered.

days

30

0.1 " +

"

"

0.1 "

Typical severe infection, with scrotal and ear lesions; recovered.
Typical severe infection; died in
10 days.

* 10 cc. heart blood were mixed with 10 cc. citrate solution; plasma collected
after centrffugafion.
Of the fourteen other guinea pigs vaccinated on the same day with the samematerial, two were tested after the same interval (30 days) by injection of virus;
both resisted infection with 100 ~.L.D. of the same virus as used in the above experiment. The remaining twelve animals were tested at intervals of 4 to 7½ months
with 1 to 1,000 ~r.L.D. of virus, and all resisted infection (see Table V).
of virus) (Table VI). I t is evident, therefore, t h a t the q u a n t i t y ofi m m u n e s e r u m m a y be liberally increased if the m i x t u r e is to be usedfor vaccination.
T h e question arose whether the i m m u n i t y which developed a f t e r
t h e inoculations of neutral or superneutral m i x t u r e s m i g h t n o t bem e r e l y a passive i m m u n i t y , due to the introduction of the i m m u n eserum.
T h i s p o i n t was decided b y the e x p e r i m e n t recorded in
T a b l e V I I which showed t h a t the inoculation of 1 cc. of a powerful

620

ROCKY

MOU2ffTAIN S P O T T E D

FEVER.

II

immune serum does not protect a guinea pig from subsequent infection 12 days or later; hence the immunity observed in guinea pigs 2
weeks or longer after the inoculation of neutral or superneutral
mixtures cannot be ascribed to a passive immunity brought about by
the antiserum introduced with the virus.

Protective Power of the Blood of Vaccinated Guinea Pigs.
From the theoretical point of view it was interesting to determine
the neutralizing power of the blood of a guinea pig which had previously been inoculated with a neutral mixture of virus (CooperBarlow strains, 1 cc.) and immune serum (No. 28, 1 cc.). Blood waswithdrawn from the heart 30 days after the time of vaccination. The
citrated plasma was separated, and 1, 0.2, and 0.1 cc. were tested
against 10 ~.r..I). of the virus (Cooper strain, 0.1 cc.). The largest
quantity, 1 cc., failed to neutralize the virus completely, but the
animal recovered, while 0.2 and 0.1 cc. had no protective effect (Table
VIII).
Fourteen other guinea pigs, vaccinated at the same time, and tested
after the same period by injection of 100 ~.L.D. of virus, all resisted theattempt to infect them. Hence the vaccinated guinea pigs are able
to neutralize a far greater quantity of virus in vivo than the low
neutralizing titer of their blood would lead us to expect.

Effect of 1tearing and of Age upon the Immunizing Properties of Neutral
Mixtures of Virus and Immune Serum.
The strikingly effective protective value of a neutral or superneutral
mixture of virus and immune serum against experimental spotted feverin guinea pigs suggested the possibility of applying the principle tohuman beings. The prospect of sero-vaccination, under the mostrigid quantitative supervision of the virus and immmae serum, ispromising; nevertheless, it seems important, for the time being atleast, to devise some method in which the virus in the mixture is killed,
but which still gives a vaccine of definite protective value.
In Tables IX and X are recorded the results of experiments in which
guinea pigs were inoculated with neutral or superneut.ml mixtures ofvirus and immune serum which had either been heated to 60°C. forH I D E Y O NOGUCHI

621

20 m i n u t e s in a w a t e r b a t h or p r e s e r v e d in t h e refrigerator (4°C.) for
32 d a y s . One s e t of a n i m a l s w a s i n o c u l a t e d w i t h p o r t i o n s of t h e
T A B L E IX.
Effect of Heating upon the Neutral Mixtures of Virus and I m m u n e Serum.

October 5, 1922. Virus: citrated guinea pig blood, Cooper and Barlow
strains (1 co. contained 100 ~r.L.D.), 1 part. Immune serum: mixture of serumfrom several immune rabbits, 1 part.
I m m u n i t y test.
Amount.

Result of vaccination.

' Interval
Tested against.

Degree of protection.

Unheated,CC.

2

No reaction.

18 days.

18
2 mos.

10,000 I~',L,D,
10,000 "100 "Complete.
Died of secondary infection.

Controls (normal rabbit serum+ virus).
2
Typical infection; diedin 11 days.
2
Typical infection; diedin 11 days.
Heated to 60° C. for 20 minutes.
2
2
2

No reaction.
"
"
"

"

18 days.
18 "2 mos.

10,000,J.L.D.
10,000 "100 "Mild fever; recovery.
"""
Died of secondary infection.

Controls (heated virus alone).
No reaction,[ 18 days,

10,000 ~,T.,D,
10,000

"

100

"

"

"

] 18-"""

2 mos.

Typical infection; died in
15 days.
Typical infection; died in
12 days.
Typical infection; died in
13 days.

freshly p r e p a r e d m i x t u r e ; a n o t h e r set received a p o r t i o n of t h e s a m e
m i x t u r e , w h i c h h a d b e e n p r e v i o u s l y h e a t e d ; t h e r e m a i n d e r of t h e
m i x t u r e w a s p l a c e d in t h e refrigerator a n d i n o c u l a t e d 32 d a y s later.

622

ROCKY MOUNTAIN S P O T T E D FEVER.

II

Suitable controls (virus m i x e d w i t h n o r m a l r a b b i t serum, a n d v i r u s
alone) a c c o m p a n i e d each of the t h r e e sets of experiments.
TABLE X.
Effect of Age u p o n the N e u t r a l M i x t u r e s of V i r u s and Immul~e S e r u m .

Portions of the mixtures of virus and immune serum used on Oct. 5, 1922
(Table IX) were kept in the refrigerator at 4°C. until Nov. 6, 1922 (32 days).
I m m u n i t y test.
Amount.

Result of vaccination.

Interval I
after
mcination.

Tested against.

Degree of protection.

6 days100 M.L.D.

Typical infection; died in
15 days.
Irregular fever (secondary
infection).
Suspicious febrile reaction
but no lesions; died in
22 days of secondary
infection.
No infection.

cG,

2

No reaction.

1 too.

100

¢¢

¢4

6 days,

100 "

"

"

5 mos.

30

2
1

1

"

"

Controls (normal rabbit serum + virus).
2
1

Secondary infection.
No reaction.

[[ 16 days.

100 x(.L.D.

Typical infection; died in
13 days.

Controls (virus alone).
No reaction.

Suspicious febrile reaction; died in 16
days; spleen enlarged.
No reaction~

1 mo.

100 ~.L.D.

16 days.

100 "

i mo.

100 "

16 days.

100"

Typical infection; died in
19 days.
Typical infection; died in
14 days.

Typical infection; died in
15 days.
Typical infection; died in
10 days.

E x a m i n a t i o n of t h e results shows, first t h a t t h e m i x t u r e w a s supern e u t r a l , since t h e r e w a s no evidence of infection in a n y of t h e a n i m a l s
i n o c u l a t e d w i t h t h e u n h e a t e d fresh m i x t u r e . T w o of these a n i m a l s

HmEYO NOGUC'HI

623

were tested 18 days later against 10,000 M.L.D. of virus, the other 2months later against 100 M.L.D., and both resisted infection. The
latter animal died of intercurrent infection 3 weeks after the dateof the iromunity test. Normal rabbit seru n had, of course, noneutralizing effect on the virus, and the two guinea pigs receiving the
mixture of normal serum and virus died after 11 days.
Of the three animals which received the heated mixture of virus andhn nune serum, two, which were tested 18 days later against t0,000
M.L.D. of virus, had a mild febrile reaction but showed no lesions; theother, which was tested 2 months later against 100 M.L.D., died of intercurrent infection after 12 days; there were no specific pathologicalfindings, and if the animal had a spotted fever infection, it must havebeen very mild. There is a great contrast between these results andthose in the guinea pigs inoculated with the heated mixture of virus
and normal rabbit serum, all of which died of typical infection.
Of the animals inoculated with the aged mixture, two died whentested 16 days later; one had a typical spotted fever infection, theother probably succumbed to secondary infection while recoveringfrom mild spotted fever. The third, tested 1 month later, had irregular fever, but no symptoms of spotted fever. Of the animals which
received the aged mixture of virus and normal rabbit serum, one diedin 13 days of typical infection, the other was lost by intercurrent
infection before the ti ne of test. The aged virus alone produced
neither infection nor immunity in the four animals receiving it; thetwo tested 16 days later died in 10 and 14 days, respectively, and thetwo tested after 1 month died in 19 and 15 days.
The contrast between the effect of fresh unheated and heatedmixtures is again strikingly brought out in Table XI, in which arepresented the results of the inoculation of smaUer quantities of thesuperneutral mixture of virus and immune serum; one series of animals
receiving respectively 1, 0.1, 0.01, and 0.001 cc. of the unheated,
another the same quantities of the heated mixture. The injection of
the heated mixture definitely modified the course of a subsequent test
infection, although complete immunity was not induced even in a doseas large as 1 cc.; the unheated mixture conferred protection in a doseof 0.1 cc., and even in the animal receiving 0.01 cc. the infection was notfatal.

624

ROCKY MOUNTAIN SPOTTED ]?EVER.

II

These findings confirm the earlier experiments establishing theeffectiveness of fresh unheated neutral mixtures of virus and immune
serum in producing immunity in guinea pigs. The uselessness ofheated or aged virus, alone, or with normal rabbit serum, is definitelydetermined. The presence of a definite immunizing property in the
TABLE XI.

Effect of Superneutral Mixtures of Virus and Immune Serum in Various Amounts.
Virus: guinea pig serum, 1 cc., representing 1,000 ~.L.D.
Immune serum: rabbit, 1 cc., representing ten times the quantity necessary toneutralize 1,000 M.L.D.
Amount.

Result of vaccination. [ ResultagainstOf
immunitYl0~s. T..test'vof34virus.
.
dayslater,

Unheated.
6C.

[¢.rus Soo ~.r.D.
1(serum 0.5 ec.

No reaction.

No infection.

[virus 50 ~r.r.D.
0.1 (serum 0.05 cc.
virus 5 XC.L.D.
0.01 ~semm 0.005 cc.
O_ ,[virus 0.5 ~.r.D.
"bX/l~sermn 0.0005 cc.

Severe infection; recovery.
Definite mild febrile No infection.
reaction.
Heated.

1

No reaction.

Mild infection; no lesions; recovery.

0.1

Moderately severe infection; recovery.

0.01
0.001

Severe infection; recovery.
Typical infection; died after
14 days.

n e u t r a l m i x t u r e which h a d b e e n h e a t e d to 56-60°C., a n d also, thoughin less degree, in the n e u t r a l m i x t u r e k e p t a t 4°C. for 32 days, indicatesthe p r o b a b l e effectiveness of mixtures t r e a t e d in this w a y for p r o p h y lactic vaccination in m a n , n o t w i t h s t a n d i n g t h a t these p r e p a r a t i o n sfall m u c h below the fresh m i x t u r e s in immunizing titer.

}IIDEYO NOGUC}II625

GENERAL CONSIDERATIONS.
From the findings outlined,itappears that the immunizing propertiesof the Rocky Mountain spotted fever virus are almost, if not totally,
removed by heating to 60°C. for 20 minutes or by prolonged preservation at a low temperature. On the other hand, the immunizing power
remains fully preserved when the virus is mixed with immune serumin neutral or superneutral proportions, notwithstanding the fact thatsuch mixtures are completely devoid of infecting power. Moreover,heating of such mixtures to 56-60°C. for 20 minutes, or prolonged
preservation at refrigerator temperature, does not altogether destroy,
though it markedly reduces, the immunizing power. Since normal
rabbit sermn does not in any way influence or retard the rapid disappearance of immunizing properties from the virus under similar
conditions (heating or aging), the persistence of immunizing power in
the neutral or superneutral mixtures appears to be due to a specificreaction which takes place between the virus (antigen) and the immune
serum (antibody).
For the purpose of inducing artificial immunity in man, the heatedneutral mixtures of spotted fever virus and a highly potent immune
serum may be used. The serum of infected guinea pigs is preferable
to that of rabbits as the source of the virus, a high degree of virulence
being more readily and uniformly maintained in the guinea pig.
Immune serum from hyperimmunized rabbits is readily prepared andis capable of attaining a high degree of potency3 These two factors,
highly virulent strains of the virus, and a powerful immune serum,
are essential to the production of an effective sero-vaccine. The
superneutral mixture is prepared as follows:
Blood is drawn from infected guinea pigs at the height of fever(4th to 5th day) and the clear serum separated from the clot. 3 The
titer should be such that i cc. contains 100 ~.L.D. of virus. By poolinga number of serums (after first making sterility tests of the individual
serums to insure absence of secondary infection), virus of uniform
8 It is imperative to determine by culture methods that the blood is free fromany secondary bacterial infection, especially that caused by members of theparatyphoid group of bacilli, which induce febrile and splenic reactions strikingly
similar to the symptoms shown by guinea pigs on the 4th and 5th days of spotted
fever infection.

626~ROCKY MOUNTAIN S P O T T E D F E V E R .

IIvirulence can be obtained in large quantity. Immune rabbit serum,of a titer such that 0.1 cc. will neutralize 100 M.L.O., is mixed with thevirus in ten times the neutralizing dose. A less potent immune serum
is not suitable, because an inconveniently large quantity would be
required. The mixture is heated to 56°C. for 20 minutes in a water
bath and preserved in the refrigerator until used. Two or moreinjections of 1 cc. df the mixture may be given subcutaneously.
The procedure just outlined is a preliminary one only. The ultimate
object of these experiments is a method in which the active (unheated)sero-vaccine may be safely applied to human vaccination.
SUMM&RY.

Freshly prepared mixtures of spotted fever virus and immune rabbit
serum in neutral or superneutral proportions confer complete immunity on guinea pigs.
The mixtures undergo a considerable loss in immunizing power whenheated to 60°C. for 20 minutes, but are still capable, if used in sufficientquantity, of conferring a degree of imTnunity on the vaccinated animalsuch that a subsequent experimental infection is rendered less severe
and non-fatal.
Unheated mixtures which had been preserved in the refrigerator at
4°C. for a period of 32 days still retained a certain degree of immunizing property.
The virus alone, or mixed with normal rabbit serum, when allowed
to die out by prolonged preservation at refrigerator temperature, or
when killed either by heating at 60°C. for 20 minutes or by chemicals(chloroform, ether, xylene) does not induce immunity in guinea pigs.