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Journal of Experimental Medicine

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IMMUNITYSTUDIES OF ROCKY MOUNTAIN
FEVER.

SPOTTED

I. USEFULNESS OF IMMUNE SERUM IN SUPPRESSING AN IMPENDING
INFECTION.
BY HIDEYO NOGUCHI, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, November 6, 1922.)

Rocky Mountain spotted fever has been for the past 20 years the
subject of study by various workers, including members of the UnitedStates Public Health Service. The discoveries of Wilson andChowning,' McCalla,2 King, 3 Ricketts, 4 Maver, 5 Moore,' Cooley,7
1 Wilson, L. B., and Chowning, W. M., The so-called "spotted fever" of the
Rocky Mountains, J. Am. Med. Assn., 1902, xxxix, 131; Studies in Piroplasmosis
hominis. "Spotted fever" or "tick fever" of the Rocky Mountains, J. Infect.

Dis., 1904, i, 31.
2 McCalla, L. P., Direct transmission from man to man of the Rocky Mountainspotted (tick) fever, Med. Sentinel, 1908, xvi, 87.
3 King, W. W., Experimental transmission of Rocky Mountain spotted fever bymeans of the tick. Preliminary note, Pub. Health Rep., U. S. P. H., 1906, xxi, 863.
4 Ricketts, H. T., The transmission of Rocky Mountain spotted fever by thebite of the wood-tick (Dermacentor occidentalis), J. Am. Med. Assn., 1906, xlvii,
358; Further observations on Rocky Mountain fever and Dermacentor occidentalis,
1067; The r6le of the wood-tick (Dermacentor occidentalis) in Rocky Mountainspotted fever, and the susceptibility of local animals to this disease-a preliminary report, 1907, xlix, 24; Further experiments with the wood-tick in relation
to Rocky Mountain spotted fever, 1278; Observations on the virus and meansof transmission of Rocky Mountain spotted fever, J. Infect. Dis., 1907, iv, 141.
5 Maver, M. B., Transmission of spotted fever by other than Montana andIdaho ticks, J. Infect. Dis., 1911, viii, 322; Transmission of spotted fever by thetick in nature, 327.
6 Moore, J. J., Time relationships of the wood-tick in the transmission ofRocky Mountain spotted fever, J. Infect. Dis., 1911, viii, 339.
7 Cooley, R. A., Preliminary report on the wood tick. Dermacentor sp. Sixth
Annual Report of the State Entomologist of Montana, Montana Agric. Coll. Exp.
Station, Bull. 75, 1909, 95; Tick control in relation to the Rocky Mountain spotted
fever, Montana Agric. Coll. Exp. Station, Bull. 85, 1911.
383

384

ROCKY MOUNTAIN SPOTTED FEVER.

I

Parker,8 and Wolbach, 9 have proved that the disease is transmitted to
man by a certain wood tick, Dermacentor venustus, which receives thevirus from infected wild rodents, principally gophers or Columbian
ground squirrels and jack-rabbits (Wilson and Chowning; Parker).
Ricketts"° was the first to transmit the disease from man to guinea
pigs and monkeys by injecting the blood drawn from spotted fever
patients and, after having fed normal ticks on the infected guinea pigs,
to reproduce the disease again in normal guinea pigs by allowing theinfected ticks to feed on the animals. The virus of Rocky Mountainspotted fever can be maintained in the laboratory for an indefinite
time by animal passage, which makes its experimental study possible.
Several strains of the passage virus1l maintained in the guinea pig
were employed in the present series of studies on immunity.
Ricketts and Gomez' 2 found that the serum from guinea pigs which
had recovered from the disease would protect normal guinea pigs
from infection when administered within 2 to 3 days after the injectionof the infecting material. The strength of the serum was such that
0.3 to 0.5 cc. was required to protect against 1 cc. of virus (1,000
infecting doses), and these investigators were unable to enhance the
8 Parker, R. R., Tick control program for 1919, Montana State Bd. Entomol.,
Circular 1, 1919; The control of Rocky Mountain spotted fever in the Bitter
Root Valley, Montana State Bd. Entomol., Circular1 (Revised), 1921. 3rd Biennial
Rep., Montana State Bd. Entomol., 1917-18, 1919; 4th Biennial Rep., Montana
State Bd. Entomol., 1919-20, 1921.
9Wolbach, S. B., Studies on Rocky Mountain spotted fever, J. Med. Research,
1919-20, xli, 1.
] 0 Ricketts, H. T., The study of "Rocky Mountain spotted fever" (tick fever?)by means of animal inoculations. A preliminary communication, J. Am. Med.
Assn., 1906, xlvii, 33.
I am indebted to Dr. L. D. Fricks, of the United States Public Health Service,
Dr. S. B. Wolbach, of Harvard Medical School, and Dr. R. R. Parker, of theMontana State Board of Health and United States Public Health Service, fortheir cooperation in supplying me with virus and ticks on numerous occasions
since 1916.
12 Ricketts, H. T., and Gomez, L., Studies on immunity in Rocky Mountainspotted fever. The preventive and curative action of a serum for spotted fever,and the inefficiency of sodium cacodylate as a curative agent for this disease inguinea-pigs, J. Infect. Dis., 1908, v, 221.

HIDEYO NOGUCHI

385

potency by repeatedly injecting an immune animal with virus. They
accordingly drew the tentative conclusion that the usefulness of theserum in human cases would be practically nil, especially since theamount required would be prohibitive when translated into terms ofhuman body weight. Ricketts' work was not completed, however,and there seem to be no experimental data to indicate how a serum of
this kind might be developed so as to be of practical value. The purpose of the experiments recorded here was to study this problem andif possible to produce an immune serum of at least sufficient potency
to be useful when the time of infection is definitely known and theinjection of serum can be made soon after the inoculation of the virus,
as when a laboratory worker is bitten by an infected tick or accidentally
inoculates himself with an instrument charged with infected material.
Although the serum of guinea pigs which have passed through asevere infection has a degree of neutralizing power when injected
at the same time as the virus, it is not strong enough to check thecourse of the infection when introduced 24 to 48 hours later. In thepresent series of experiments, however, it has been found that theneutralizing power of the serum is considerably increased by one
or two more subsequent inoculations of the virus, in citrated blood,
into the convalescent guinea pig, and sera prepared in this way arecapable of modifying the course of the disease into a non-fatal one or,in some instances, of suppressing the infection completely, provided
the serum is administered during the incubation period.
The small size of the guinea pig precludes the possibility of utilizing its immune serum for human cases, but the principle developed
from the experiments with the serum of guinea pigs suggested thatlarger animals might be capable of furnishing an equally potent
immune serum in correspondingly greater amounts.
Ricketts and Gomez,' 2 and later Heinemann and Moore,13 reported having
infected horses by injecting large quantities of virus from guinea pigs or monkeys.
Although the horses showed some fever on the 3rd or 4th day after inoculation, it
never reached 1040F. and usually lasted only 1 day. The serum from the horses,
however, was stated to possess some neutralizing power as compared with normal
horse serum.

1' Heinemann, P. G., and Moore, J. J., Experimental therapy of Rocky Mountain spotted fever, J. Infect. Dis., 1912, x, 294.

386

ROCKY MOUNTAIN SPOTTED FEVER.

I

EXPERIMENTAL.

The writer's experiments with horses have so far been unsuccessful. Two adulthorses were given intravenous injections of blood from infected guinea pigs indoses ranging from 20 to 50 cc. at intervals of 3 to 5 days, and a colt 6 months oldreceived similar injections of larger amounts (35 to 95 cc.) of the virus. The riseof temperature to 103-104°F. which occurred on the 3rd or 4th day after inoculation was interpreted as being due to the introduction of a large quantity of blood
corpuscles from a foreign species. The animals never showed symptoms suggestive of spotted fever, and the serum on test was found to have only slightneutralizing power for the virus; 1 cc. was necessary to neutralize about 10 M.L.D.
when administered simultaneously.
In view of the facts that adult ticks feed on horses and cattle, and that a certain
number of ticks collected at random prove to be infective, it was thought of interestto test the serum of horses which had been pastured in infected districts. Serumfrom a horse which had been pastured for several years in the Bitter Root Valley
was obtained' 4 and tested against the virus. As shown in Table I this serum(No. 1) in a dose of 3 cc. gave complete protection against 1 cc. (approximately
100 M.L.D.) of the spotted fever virus when injected simultaneously. Of the otherthree sera subsequently obtained from horses pastured in the same region, one(No. 2), which was contaminated on arrival, failed to give any protection in adose of 3 cc., while Sera 3 and 4 had definite neutralizing power, the animals receiving 3 cc. of the serum simultaneously with 100 M.L.D. of the virus recovering
after a typical febrile reaction without scrotal or skin lesions.
TABLE I.

Administration of Serum from Horses Pastured for Several Years in the Bitter Root
Valley of Montana to Guinea Pigs Inoculated with 100 M. L. D. of the Rocky
Mountain Spotted Fever Virus.
Serum No.

Date.

Amount.

Result.

1922

cc.

1

Feb. 33

1Apr. 23 G"

2(contaminatedon arrival).
3

"

25

3

Typical infection.

"

25

3

4

"

25

3

"
fever for 4 Recovered without lesions.
days.
Typical fever for 5
days.

No infection.

"

Remarks.

Tested for immunity after 40days and found to be completely immune.
Died of intercurrent infection
before test for immunity
could be carried out.
Died in 13 days.

14 The serum was obtained through the kindness of Dr. W. J. Butler and Dr.
Hadleigh Marsh, of the Montana Livestock Sanitary Board.

HIDEYO NOGUCHI387

The only animal which yields a useful serum, both in potency andquantity, is the rabbit. In the first place, the rabbit is susceptible
to the virus of spotted fever. The infection is usually non-fatal,
the febrile period lasting for 6 to 9 days, though it is sometimes shorteror longer. The scrotal skin may show the typical capillary thrombosis
and subsequent necrosis. After recovery the animal becomes completely resistant to subsequent infection, and 0.5 to 0.1 cc. of theserum, collected 2 weeks after disappearance of symptoms, is capableof rendering non-fatal an infection produced by 100 M.L.D. of the virus,
the test animal in every instance being the guinea pig. By anotherintravenous injection of virulent blood, e.g. of 4 cc., or 400 M.L.D.,
the serum can be raised in potency five- to tenfold, and in some instances to such a titer that 0.1 cc. will neutralize 1,000 M.L.D. The
reinforcing injection is given 2 weeks after complete recovery of theanimal, and the immune serum is drawn 9 days later. A serum,
whether convalescent or reinforced, is sufficiently strong for practicaluse when it is capable in a dose of 0.1 cc. of neutralizing 100 M.L.D.
of the virus. As will be seen from the results recorded in Tables II
to IV, the immune rabbit serum prevents the development of theinfection or renders it non-fatal when administered within the period
of incubation-the earlier the administration of the serum the more
complete the suppression of the infection.
Male guinea pigs of 500 gm. body weight were used. The virus '"consisted of the citrated blood from the heart of guinea pigs on the
4th or 5th day of fever and was inoculated subcutaneously. 1 cc.,equivalent to 100 M.L.D., was given in Experiment 1, 0.1 cc. (10 M.L.D.)
in Experiment 2, and 0.01 cc. (1 M.L.D.) in Experiment 3. Eighteen
guinea pigs were used in each series, two animals being inoculatedwith the immune serum immediately after the injection of the virus,
and two at the end of 24, 48, 72, and 96 hours, and 5, 6, and 7 days.
The remaining two animals served as controls without serum. The immune serum used was a mixture of sera from several immunized rabbits and was given in a dose of 1 cc. in Experiments 1 and 2 and 0.1
cc. in Experiment 3. The titer of the pooled serum was such thatwhen given simultaneously 1 cc. completely neutralized 100 M.L.D.,
15 These experiments were carried out with a strain furnished by Dr. Parker.

TABLE II.

Experiment 1. Administration of Immune Rabbit Serum to Guinea Pigs Inoculatedwith 100 M. L. D. of the Rocky Mountain Spotted Fever Virus.
Amount of serum, 1 cc. uniformly.
Guinea pig No.

1A

Interval before serum
administration.

Symptoms before serum
administration.

Immediately after None.

Symptoms after serum
administration.

None.

inoculation.

1B

Immediately after
inoculation.
hrs.

2A
2B
3A

24
24
48

3B

48

4A

72

4B

72

5A

96

5B

96

Temperature rose to 104.50
F. in 7 days; remained
high for 24 hrs. Recovered.
High fever developed in 9
days; lasted for 4 days.
No
scrotallesions.
Recovered.

High fever on 6th day; remained for 4 days. No
scrotal lesions.
Recovered.
c¢High fever for 3 days after
5th day. No scrotallesions. Recovered.
High fever continued for
5 days. No scrotallesions. Recovered.
0Temperature 105 F. in High fever in 7 days,
morning.
lasting 48 hrs. No
scrotallesions. Recovered.

days

Feser for 2 days.

6A

5

6B

5

High fever for a day.

7A

6

..
" (105-107 °F.) for 2 days.

7B

6High fever for 2 days.

388

No abatement of temperature. Typical course.
Died in 16 days.
Fever continued at 105106.5°F. for 8 days.
Died in 17 days.
No fall in temperature.
Typical course. Diedin 13 days.
No reduction in fever.
Typical course. Died in
13 days.

389

HIDEYO NOGUCHITABLE II-Concluded.
Guinea pig No.

Interval before serum
administration.

Symptoms before serum
administration.

Symptoms after serum
administration.

High fever for 3 days.

Apparently no effect on
the course of infection.
Died in 13 days.
No beneficial effect. Typical course. Died in 12
days.

days

8A

7

8B

7

"

"

"

3

"

Controls without serum.
9A
Temperature 105 0F. after 96 hrs. and remained high for 5 days; fell tosubnormal on the 11th day. Scrotum, ears, and feet showed typicallesions. Died in 12 days.
9B
Rise of temperature to 104.5°F. in 96 hrs.; continued high for 7 days.
Died in 13 days.

0.1 and 0.01 cc. modified the infection to a non-fatal one, while
0.001 cc. exerted no influence upon the course of the infection. The
serum was given subcutaneously.
As Table II shows, 1 cc. of the serum, given within 24 hours fromthe time of introduction of a massive amount (100 M.L.D.) of the virus
into the body of the guinea pig, is capable of suppressing the infection;
when given later, but before 96 hours (near the time of onset of thedisease), this quantity does not suppress the infection but renders
it non-fatal. After 5 days, that is when the disease is already manifest, no benefit seemed to be derived from administration of theserum, the animals dying within 12 to 17 days (the controls diedwithin 12 to 13 days).
The results in the second series (Table III) are similar, except
that, owing probably to the smaller quantity of the virus used (10M.L.D.), the infection was completely suppressed in all animals which
received the serum within the first 48 hours. All those which received
it after 48 hours, but within 96 hours, survived, after passing througha mild infection without any scrotal or other cutaneous lesions.
Administration of the serum later than 5 days invariably failed toprevent a fatal outcome, the animals dying just as the controls did.
In the third series (Table IV), in which only 1 M.L.D. was used to
infect the animals, the protective effect of the immune serum wasTABLE III.

Experiment 2. Administration of Immune Rabbit Serum to Guinea Pigs Inoculatedwith 10 M.L.D. of the Rocky Mountain Spotted Fever Virus.
Amount of serum, 1 cc. uniformly.
Guinea pig No.

10A
1OB

Interval before serum
administration.

Symptoms before serum
administration.

Immediately after None.
inoculation.
Immediately after
inoculation.
hrs.

11A
11B
12A
12B
13A

24
24

13B

72

14A

96

14B

96

Symptoms after serum
administration.

None.

it

48
48
72

High fever afte 5 days;
lasted only 3 days.
Recovered.
High fever after 5 days;
lasted 4 days. Recovered.
High fever developed in 7
days; lasted 2 days. No
scrotal lesions. Recovered.
High fever in 7 days;
lasted 5 days. No scrotal lesions. Recovered.

days

15A

5

15B

5

16A

6

16B

6

17A

7

17B

7

First sign of fever (1050 Fever continued for 7 days.
Typical infection. Diedin 12 days.
High fever continued for
Beginning of fever.
8 days. Died in 13days.
. high fever. Fever of typical course.
Died in 13 days.
Fever remained high forHigh fever for 24 hrs.
" high fever"
7 days. Died in 14
days.
No influence on the courseof the infection. Diedin 13 days.
cc48
Fever for 5 days. Typical course. Died in 12
days.

Controls without serum.
18A
Temperature 105.20 F. in 5 days; remained high for 7 days. Died in
13 days.
18B
Temperature 105 0F. in 5 days; remained high for 6 days. Died in 12
days.
390

391

HIDEYO NOGUCHITABLE IV.

Experiment 3. Administration of Immune Rabbit Serum to Guinea Pigs Inoculatedwith 1 M.L.D. of the Rocky Mountain Spotted Fever Virus.
Amount of serum, 0.1 cc. uniformly.
lGuinea pig No.

19A
19B

Interval before serum
administration.

iSymptoms before serum Symptoms after serum administration.
administration.

Immediately after None.
inoculation.
Immediately after
inoculation.

None.

hrs.

20A
20B
21A
21B
22A
22B
23A
23B

24
24
48
48
72
72

96
96

i'

4'

'C
C~
cc
C'
i
cc

Fever in 7 days; lasted 5 days.
Recovered.

days

24A

5

24B

5

25A

6

25B

6

26A

7

26B

7

Fever in 7 days; lasted 48 hrs.
Recovered.
Temperature 105 ° Fever lasted for 8 days. Typical scrotal lesions. Died
F. in the morningin 16 days.
Temperature 1060 Fever remained high for 7 days.
F. in the morning. Typical infection. Died in
14 days.
Temperature 106 0F. in 9 days;
None.
lasted 48 hrs. Recovered.
cc
Fever in 9 days; remained high
3 days. Recovered.
Temperature 105.5° Fever and scrotal lesions typF. for 24 hrs.
ical. Died in 17 days.

Controls without serum.
27A
Temperature rose to 105.5 0F. in 5 days; lasted for 9 days. Scrotal skinnecrotic; ears hardened. Died in 18 days.
27B
Temperature 105 0 F. in 7 days; lasted for 8 days. Scrotal lesions typical.
Died in 16 days.

much more pronounced than in the first two series. Here the infection was completely suppressed when 0.1 cc. of the serum wasgiven within 72 to 96 hours. In one of each set of two animals treated392

ROCKY MOUNTAIN SPOTTED FEVER.

I

with the serum after 5, 6, and 7 days respectively, the infection was
mild, undoubtedly because the serum was given before the animals
showed any fever, while they were still in the incubation period,
which is decidedly longer in the case of 1 M.L.D. than when 10 or 100
M.L.D. are inoculated.
DISCUSSION AND SUMMARY.

From the results of the experiments presented it is evident that inguinea pigs an early administration of immune rabbit serum will
suppress the infection; that is, if it is given within the period of incubation, the effect being proportionately greater the earlier the serum
is administered. Almost no beneficial effect is observed when theserum is given after the onset of the disease. In the animals inoculated with 10 to 100 M.L.D. the incubation period is shorter than when
1 M.L.D. is injected; nevertheless 1 cc. of the immune serum saved theanimals as late as 96 hours from the time of the introduction of thevirus into the system. When administered within 24 hours in thecase of 100 M.L.D. and within 48 hours in the case of 10 M.L.D., theserum completely neutralized the virus, and the animals escaped
infection altogether. On the other hand, the same quantity of theserum only modified the infection into a non-fatal one when given
a day or two later. In the animals which were inoculated with
1 M.L.D. the incubation period was a day or two longer, and the neutralizing effect of the serum was much more powerful. Here animals
were saved as late as 5, 6, and 7 days and with a much smaller quantityof the serum (0.1 cc.).
As to the usefulness of such an immune serum in human cases,
the relative susceptibility of man and the guinea pig must first be
considered. In a large number of experimental infections carriedout with guinea pigs in the past 6 years almost never has a naturally
refractory animal been encountered. The mortality is nearly 80
per cent with most strains, although as low as 50 per cent with some.
The strain used in the present study caused death in nearly 80 per
cent of the animals. Hence the susceptibility of guinea pigs is atleast as great as that of man, in whom the mortality in the Bitter
Root Valley is estimated to be about 70 per cent.'?9

HIDEYO NOGUCHI393

The relative length of the incubation period in guinea pig and in
man is another point which requires analysis. In guinea pigs it
varies somewhat according to the number of passages, being as short
as 3 days when 100 M.L.D. or more of an adapted virus are inoculated.
On the other hand, when the infection is the result of 1 M.L.D. or thebite of an infected tick, the incubation period is much longer, being
5, 6, or 7 days in the former and 7 to 8 days in the latter instance, aswith the present strain. In man the infection is brought on by thebite of an infected tick, and the period of incubation varies from 3
to 10 days but is usually 7 days; i.e., it is about the same as in guinea
pigs infected with 1 M.L.D. Hence we may regard the susceptibilityof man and the guinea pig as nearly equal.
The final point to be considered is the quantity of the immune serumthat may be recommended for use in human cases. To prevent theinfection in a guinea pig weighing 500 gm., 0.1 cc. of the serum was
sufficient. This quantity protected the animal against 1 M.L.D. evenas late as 5, 6, or 7 days. Calculated on this basis, 16 cc. of the serum
would be required for a man weighing 80 kilos (about 160 pounds); thatis, 16 cc. of an immune rabbit serum, administered before onset of thedisease, should theoretically be sufficient to save a man of average
weight against an infection brought about by the bite of an infected
tick or by a laboratory accident. It would probably be best toadminister the serum intravenously. The titer of the immune serum
should be previously determined in guinea pigs, and 1 cc. should
neutralize 100 M.L.D. completely and 0.1 and 0.01 cc. render the infection non-fatal. Such a serum is easily produced in rabbits (a rabbit
weighing 2,500 gm. will yield 50 to 60 cc. of the serum) and probably
will remain active a year or longer when kept at refrigerator
temperature.
CONCLUSIONS.

A potent immune serum against the virus of Rocky Mountainspotted fever can be produced in the rabbit.
By early administration of such an immune serum the virus introduced into the guinea pig can be prevented from multiplying
and causing fatal infection. When given within the period of incubation the suppression of the infection is assured, but after the onsetof the disease the serum has no beneficial effect.

394

ROCxKY MOUNTAIN SPOTTED FEVER.

I

In view of the comparative susceptibility of man and the guinea
pig it is recommended that in every instance when the bite of a tickgives reason to suspect a possible infection with spotted fever, or
when a person accidentally inoculates himself with the virus, about
0.2 cc. of the serum per kilo of body weight (or 0.1 cc. per pound) be
injected immediately, preferably intravenously. For the average
adult about 16 cc. should be given.