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Studies on Rocky Mountain spotted fever online

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ture of guinea-pig, 104.4; January 5, temperature of guinea-pig, 106;
January 6, temperature of guinea-pig, 105.6, killed.

Autopsy showed typical lesions of spotted fever, and inoculation of
the heart's blood reproduced the disease in two other guinea-pigs from
which a third series of guinea-pigs were infected; each animal reacted
characteristically and showed typical lesions.

Result: Tick XXVIII. proved to have transmitted the
virus of Rocky Mountain spotted fever. Tick XXVIII. dis-
sected January 23, 191 7.

The organisms were found in smear preparations of salivary
glands, Malpighian tubes, gut wall and muscles.

Tick XXXIII. and Tick XXXIV. Two unfed D. venus-
tus 9 s. Fed with others May 22 to May 24, 1917, on one



ROCKY MOUNTAIN SPOTTED FEVER. 97

of two guinea-pigs inoculated with one cubic centimeter of
blood May 18, 1917, from a human case of spotted fever at
Hamilton, Mont. (Case III., John Lake.)

Temperatures of Case III. guinea-pig: May 18, inoculated; May 19 •
103.2; May 21, lOi; May 22, 105, ticks attached; May 23, 104.9;
May 24, 105, scrotum swollen and red, ticks removed; May 25, killed.

This guinea-pig was not autopsied. The companion inoculated at the
same time ran a similar course of temperatures and at autopsy, May 25,
showed typical lesions of spotted fever.

These two ticks were attached in a capsule on June 2, 1917, to a normal
guinea-pig, recorded as Case III. Guinea Pig 3. Removed June 11, 1917.

Record of Case III. Guinea Pig 3. — Temperatures: June 2, ticks
attached; June 4, 102, one tick feeding; June 5, 103, both ticks feeding;
June 6, 102.2; June 8, 101.8; June 9, 101.2; June 11, 101.6, ticks
removed; June 12, 102.4; June 13, 101.6; June 14, 102; June 15, 103.4;
June 16, 104; June 18, 104.6; June 19, 105; June 20, 105; June 21,
103; June 22, dead.

Autopsy showed typical lesions of spotted fever.

Result : One or both of Ticks XXXIII. and XXXIV. proved
to have transmitted the virus of Rocky Mountain spotted
fever.

Ticks XXXIII. and XXXIV. dissected June 29, 1917.

Both ticks showed the organisms in marked abundance in
all tissues in serial sections and in the smear preparations of
salivary glands, gut, Malpighian tubes and muscles. (Fig. 30.)

Summary: Ten proved infective adult ticks contained the
parasites of Rocky Mountain spotted fever.

(2) Ticks infected as nymphs, some of which were proved
to be infective in the adult stage.- — The " flat," i.e., unfed,
nymphs used in this series of examinations for the presence
of the spotted fever organisms were received from W. V. King
in June, 1917. These nymphs were raised by Dr. King from
eggs and reached the nymphal stage in July, 1916. Seven
which fed on a normal guinea-pig (see first control series,
Table II.) did not infect. On July 2, 1917, 200 were placed
in a jar with Guinea Pig 19, Hayes strain. On July 3, 127
more nymphs were added, making a total of 327. On July 5 ,
the guinea-pig with the feeding nymphs was transferred to a



98 WOLBACH.

new jar and the first one sterilized. On July 9 many fully
engorged nymphs had dropped, and on July 11, 140 fully
engorged nymphs were collected from the jar. The majority
of these nymphs molted during the first week in August, and
the adults reserved for future experiments were placed in a
cold room, in a slightly moistened atmosphere, temperature
7° C. to 10° C.

The temperatures of this guinea-pig were not taken because
of the danger of losing some of the nymphs. It was killed
July II, 191 7, and showed the typical lesions of spotted fever.
The inguinal lymph nodes were enlarged and red. The spleen
was treble normal size, deep red in color; the capsule was
covered with a thin, translucent fibrinous layer. The testes
were adherent to the tunica, with hemorrhages into the polar
fat. The cremasteric muscles were dry, dark-red in color
and adherent. The skin of the scrotum was necrotic and the
subcutaneous tissues of the scrotum were oedema tous and
dark-red in color. The other tissues of the guinea-pig were
negative.

Experiments to prove the infectivity of the ticks fed as
nymphs on Hayes Strain Guinea Pig 19. — I. August i"! ,
191 7, placed three female and three male ticks of this series
in a wire gauze capsule upon a normal guinea-pig subse-
quently recorded as Hayes Strain Guinea Pig 44. (See Chart
4-)

Record of Hayes Strain Guinea Pig 44. August 27, ticks attached;:
August 28, loi, none feeding; August 29, 100, all ticks feeding..
Temperatures: August 30, 100; August 31, 101.6; September i, 102.7;,
September 3, 105.6; September 4, 106, removed ticks; all have fed mod-
erately; September 5, 105.4, killed guinea-pig for inoculations.

Autopsy showed the typical lesions of spotted fever, and from this
guinea-pig the strain was maintained. (See Chart 4, page 59.)

Result: Six ticks fed as nymphs on Hayes Strain Guinea
Pig 19 proved to have transmitted the virus of Rocky Moun-
tain spotted fever.

n. August 23, 1 91 8. Placed two female »and two male
ticks of this series in a wire gauze capsule upon a normal;



ROCKY MOUNTAIN SPOTTED FEVER. 99

guinea-pig, subsequently recorded as Hayes Tick Guinea Pig
of August, 1918.

Record of Hayes Tick Guinea Pig of August, 1918. Temperatures:
August 23, 102.4, ticks attached; August 24, 101.6, three ticks feeding;
August 25, loi.i, two ticks are feeding; August 26, 101.6, all ticks are
feeding; some have passed feces; August 27, 100; August 28, 104; August
29, 102.6, removed ticks; all have fed moderately; August 30, 105.3,
inoculated Guinea Pigs i and 2 with blood taken by heart puncture;
August 31, 106.6, killed.

The autopsy showed the typical lesions of spotted fever, and Guinea
Pigs 3 and 4 inoculated with the heart's blood were used to maintain the
strain. (See Chart 5, page 60.) On Guinea Pig 13 of this series other
ticks reared from eggs of a tick (Tick A) were infected and Hayes Tick
"A " strain established. (See Chart 5, page 60.)

Result : Four ticks fed as nymphs on Hayes Strain Guinea
Pig 19 proved to have transmitted the virus of Rocky Moun-
tain spotted fever.

HI. On August 5, 1918, placed five female ticks of this
series in a wire gauze capsule upon a normal rabbit (white and
black), subsequently recorded as Hayes Tick Rabbit i.

Record of Hayes Tick Rabbit i. Temperatures: August 5, 102.2,
attached ticks; August 6, 102.2, all ticks are feeding; August 7, 103,
feces in capsule; August 8, 102.4; August 9, 103.4; August 10, 105.7,
removed ticks; all have fed well (average size 5x6x2 mm.); August 11,
105.4, scrotum injected and slightly swollen; August 13, 105.2; August
14, 105.4; August 15, 104.2, killed.

The autopsy showed enlarged and pink inguinal lymph nodes, the
spleen deep red in color and moderately enlarged, and the testes injected.
All other tissues were negative. Microscopic examination of the tissues
showed the typical vascular lesions of spotted fever in the testes and skin
of the scrotum, and the parasites were found in these lesions.

Result: Five ticks fed as nymphs on Hayes Guinea Pig 19
proved to have transmitted the virus of Rocky Mountain
spotted fever.

IV. December 19, 191 8, placed two female ticks of this
series, which had also fed on Hayes Tick Rabbit i, on a normal
guinea-pig which was recorded as Hayes Tick Guinea Pig of
December, 1918.

Record of Hayes Tick Guinea Pig of December, 1918. Temperatures:
December 19, ticks attached; December 20, 103.8; December 23, .101.6;
December 24, 102.6, removed ticks which had fed sufficiently to fill out



100



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ROCKY MOUNTAIN SPOTTED FEVER. IO7

the wrinkles and pass feces; December 26, 103.6; December 27, 105.2;
December 28, 106.4, killed.

The autopsy showed typical lesions of spotted fever and from this
guinea-pig the strain was maintained for eleven generations. (See Chart
5, page 60.)

Result: Two ticks fed as nymphs on Hayes Strain Guinea
Pig 19 and again upon a normal rabbit were proved to have
transmitted the virus of Rocky Mountain spotted fever.

Summary of conditions affecting the preceding Table I. —
Seven nymphs from the same source fed on a normal guinea-
pig did not infect. (See first control series, Table II.) Of
ticks raised from one hundred and forty nymphs fed on Hayes
Guinea Pig 19, three separate lots of six, four and five respec-
tively transmitted the virus of Rocky Mountain spotted fever,
and in a fourth experiment two ticks of the five used to infect
a rabbit also infected a guinea-pig. It seems reasonable to
■conclude that the majority or all of the ticks fed as nymphs on
Hayes Guinea Pig 19 contained the virus of Rocky Mountain
spotted fever.

Summary of results in Table I. — Forty-five nymphs fed
■on Hayes strain Guinea Pig 19 were dissected and examined
for the parasite of Rocky Mountain spotted fever during a
period of forty-seven days extending from the first day after
■dropping as fully engorged nymphs through and into the
adult stage. The smear preparation technic was controlled
by selecting at random ticks throughout the series for serial
sectioning.

The parasites were found on and after the fifth day (note
influence of temperature in chart) with but one exception
(Tick LXXXVL).

The first form to appear in smears of the gut is the relatively
large pale-blue staining rod. at which time the minute intra-
nuclear forms may be found in sections of the rectal sac and
intestines.

In the early stages of digestion in the nymph many confusing
bodies are found, free and within cells of the intestinal tract.
For a while, tiny red and blue stained bodies, one or two



I08 WOLBACH.

microns in diameter, massed in epithelial cells of the intestine,
were given consideration as possible parasitic forms, as their
resemblance to Theileria was rather striking. However, these
forms in later stages of digestion lost their red and blue
staining and many became pigmented greenish. A study
of the control nymphs showed the presence of these bodies
in equal abundance, thereby absolutely disproving relation-
ship to the parasite of Rocky Mountain spotted fever. As
digestion in the tick is wholly intracellular, the most plausible
explanation of these bodies, and one supported by considerable
morphological evidence, is that they are derived from the
nuclei of ingested white blood corpuscles.

With the appearance in the intestine of the minute rods
with chromatoid granules, the large forms diminish in num-
bers and finally disappear (eight to fifteen days, according to
temperature). Still later the densely staining lanceolate
forms appear in the intestines; from about the sixteenth day
on. The period of greatest abundance of the minute rods
with chromatoid granules in smears of the intestinal tract
corresponds with the greatest abundance of intranuclear
forms in sections. Lanceolate forms, however, can be found
in sections in most organs immediately after the intranuclear
forms make their appearance.

Since the lanceolate is the only form that caii be demon-
strated in the blood of infected mammals, it is probably the
one introduced into the tick. From this densely staining
lanceolate form must come the pale-blue staining rods which
are the first indication of multiplication of a specific parasite
in infective ticks. The period of greatest abundance of the
parasite in the tick is that coincident with the greatest intra-
nuclear development, and the minute rods with chromatoid
dots. The lanceolate form is a late arrival in the tick, and
represents the completion of a fairly definite morphological
cycle, and as the lanceolate form only can be demonstrated
in the salivary gland cells and ducts of both types of salivary
gland acini in the tick, it is reasonable to suppose that the
parasite is reintroduced into mammals in this form.



ROCKY MOUNTAIN SPOTTED FEVER.



109



Confirmatory evidence of this last theory is the fact that
the lanceolate paired forms were found in large numbers in
mononuclear phagocytic cells (endothelial cells) in sections
through a tick bite made in infecting Hayes Strain Guinea
Pig 3.

(3) Control ticks. Nymphs proved non-infective. First
series. — These nymphs were raised by Dr. W. V. King from
eggs in July, 1916, in Victor, Mont. They were fed on a
normal male Guinea Pig 6, August 4 to 13, 1917, when seven
fully engorged nymphs were recovered, which had dropped
from the guinea-pig.

Temperatures of normal Guinea Pig 6 after removal of the nymphs:
August 13, 102.4; August 14, 102.5; August 15, 102; August 16, 101.4;
August 17, loi; August 18, 102; August 19, 20, lOi; August 21, 100;
August 22, loi; August 23, 102.4; August 24, 101.6; August 25, loi.

This guinea-pig was not tested for susceptibility, as it died
on August 27, together with several others, from the effects
of an exclusive cabbage diet.

Table II.



No. of Tick.



Date Dissected, Time after Dropping,
Temperature Conditions.



Smears of Gut

and Malpighian

Tubes.



Serial Sections,
All Tissues.



CIV. .
CVI. .

evil..

CVIII.
CIX. .
CXIV.

CXIX.



August 16, 3 days at 37.5° C. in incuba-
tor.

August 17, 4 days at 37.5° C.

August 20, 7 days at 37.5° C.

August 20, 7 days at 37.5° C.

August 22, 9 days at room temperature.

August 23, 9 davs at room temperature;


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