The purpose of this communication is to record the demon

THE ETIOLOGY OF ROCKY MOUNTAIN SPOTTED FEVER.*
(A Preliminary Report.)
S. B. WOLBACH, M.D., BOSTON.
(Fromt the Bacteriological Laboratory, Harvard University Aedical School.)
The purpose of this communication is to record the demonstration of a parasite in the lesions characteristic of experimental spotted fever in guinea-pigs and in monkeys. The
virus was secured in the form of infected ticks -Dermacentor andersoni, Stiles (Dermacentor venustus, Banks),
through the kindness of Surgeon L. D. Fricks of the United
States Public Health Service, to whom I express my thanks.
Two series of guinea-pigs were started from these ticks:
one from a male; the other from a female tick. In both
instances the ticks remained attached to the guinea-pigs for
over forty-eight hours. Characteristic symptoms appeared
in the original two guinea-pigs, and no serious difficulty has
been experienced in maintaining the succession. That the
disease established from these two ticks is spotted fever, as
it occurs experimentally in guinea-pigs, is certain, from the
character of the incubation period, the course of the fever,
the appearance of rash upon the scrotum, and the swelling
with hemorrhages into the skin of the scrotum; in many
instances there was swelling of the ears, with rash, and swelling of the legs followed by necroses of the paws.
The post-mortem findings, at all stages, have agreed in
every respect with those described by Ricketts and others,
both in the disease as transmitted directly from the blood of
human patients, and through the bites of infected ticks.
Many cultural tests, on a large variety of media, under varying conditions as to temperature and oxygen tension, have
proved the sterility of the heart's blood and tissues for
ordinary bacteria. The microscopic findings agree with
those described by Le Count.
* Received for publication
(121)
Feb. 23, I9I6.
122
WOLBACH.
The essential lesions of spotted fever take origin in the
vascular system. These lesions are responsible for the rash,
the edema, the hemorrhages, and the necroses observed both
in human beings and in experimental animals. In these
lesions there occurs a minute parasite, in large numbers,
having some of the characteristics described by Ricketts for
the bodies which he found in the blood of patients and
experimental animals, and in the organs and eggs of infected
ticks. Ricketts regarded these bodies as bacilli; their size
he stated to be approximately that of Bacillus influenzae.
He describes them as having the form of "two somewhat
lanceolate chromatin-staining bodies, separated by a slight
amount of eosin-staining substance." This description was
based on preparations of blood stained by Giemsa's stain, as
furnished by Griibler.
It is not possible to state conclusively that the organisms
present in the lesions of spotted fever are bacilli in the
ordinary sense of the term, because of peculiarities in their
distribution and their staining reaction, which I find to be
somewhat different from that described by Ricketts, and
markedly different from the staining reaction of most bacteria. It is my opinion, however, that these organisms are
bacilli.
They occur in the greatest numbers in the less advanced
lesions of blood vessels (both arteries and veins), in the
testicle and its appendages, in the cremasteric muscles, and
in the skin and subcutaneous tissues. They were first found
in sections. Ordinary methods of making smear preparations, and contact (klatsch) preparations from the internal
organs and lymph nodes, failed to reveal the organisms.
After their location in the tissues had been determined, it
became possible to demonstrate them in smears made from
teased preparations. They may be satisfactorily demonstrated in smears made by scraping affected tissues with a
sharp knife, held vertically.
The characteristic form is a short rod in pairs, joined end
to end; many of the rods exhibit bipolar staining; this,
ETIOLOGY OF ROCKY MOUNTAIN SPOTTED FEVER.
123
however, is only satisfactorily demonstrable in smear preparations. The organisms are found in apparently uninjured
endothelium of normal vessels, in areas of proliferated endothelium of the intima of vessels, in hyaline necrosed intima
in more advanced lesions, in apparently normal and necrosed
smooth muscle fibers of vessels with lesions, and in endothelial cells in the perivascular zones of proliferation. They
occasionally occur within endothelial cells in dilated lymphatics. They may also be found between the cells of vessels
showing lesions, and in fat tissue and edematous connective
tissue. The largest masses are seen in smooth muscle cells
of affected arteries and veins, and occasionally they occur in
enormous numbers in such cells. There is considerable variation in size. In the densely packed smooth muscle cells
they appear to be distinctly smaller and shorter than in other
locations. The organisms are often surrounded by a
slightly refractive clear zone, which is most marked when
they are seen in necrotic smooth muscle cells.
In smear preparations, stained by Giemsa's stain, granular
and lanceolate forms can be observed. The organisms
appear to be larger than in sections. They are found almost
exclusively within endothelial and smooth muscle cells. In
one instance only has an organism been found in a granular
leucocyte. I have also found them to be easily demonstrable
in thick film preparations made directly from the heart's
blood at autopsy. They stain bluish, in marked contrast to
most bacteria, which take an intense reddish purple stain.
This reddish purple coloration with the Romanowsky stains
is regarded as the chromatin staining reaction, so that I am
somewhat at a loss to understand the description " chromatin
staining " by Ricketts as applied to this organism. The
characteristic arrangement of the bacilli, end to end, and
the bipolar effect is shown in the accompanying photomicrographs. The length of the organism in tissues ranges
from one-half to one micron; in smear preparations, from
three-fourths to one and a half microns. The width is from
.2 to .5 micron.
124
WOLBACH.
The photomicrographs having been made accurately at
two thousand diameters will give the best idea of their size.
Occasionally forms are encountered which are thinner, with
tapering ends, and having small reddish stained granules or
areas in them. It is possible that these represent degeneration forms or possibly another stage. No work has as yet
been done with ordinary bacterial stains in smears. In
sections the organisms are Gram negative.
Technic of demonstrating the organisms in sections.The organisms were first found in Zenker fixed tissues,
stained by the eosin-methylene blue method of Mallory.
This staining method gives somewhat better results after
fixation in corrosive alcohol. A satisfactory method of
demonstrating the organisms is to stain in Loeffler's alkaline
methylene blue for from twelve to twenty-four hours in the
paraffin oven (55' C.) and differentiate in I-IOOO or I-2000
acetic acid solution in water. But a few seconds are required
for differentiation, after which the sections should be rapidly
dehydrated in absolute alcohol and cleared in xylol. The
best method for their demonstration, in relation to tissues, is
Giemsa's stain applied after Zenker fixation. Owing to
their peculiar staining reaction they cannot be satisfactorily
demonstrated by Giemsa's stain after alcohol corrosive
fixation. Having in mind the reversal of Giemsa's stain,
when Zenker fixation is employed, the experiment was made
and it was found that the bacilli may be readily stained and
acquire a deep blue color, thereby furnishing an advantageous contrast to the pink-staining materials in which they
are most frequently found. The Giemsa stain is applied in
the same manner as for staining after alcohol corrosive fixation and allowed to act for twelve to twenty-four hours, after
which the sections are differentiated in a methyl alcoholcolophonium mixture (fifteen per cent colophonium in equal
parts of acetone and methyl alcohol). The differentiation
takes place rapidly, and should not be carried too far. It
should be followed by a mixture of xylol (seventy per cent)
ETIOLOGY OF ROCKY MOUNTAIN SPOTTED FEVER.
125
and acetone (thirty per cent), then xylol, and sections
mounted in cedar oil.
SUMMARY.
An organism, a bacterium, having certain peculiar characteristics, may be found in large numbers in the lesions
characteristic of spotted fever in experimental animals.
These lesions are essentially proliferative in character. The
cells which respond in largest numbers to the action of the
organisms are endothelial cells. The5e accumulate in great
numbers in the vessel walls and around the vessels. They
may be seen in mitosis, in various locations, and in Jymphatics
and blood vessels. The organism corresponds in some
respects with the description given by Ricketts of bodies
which he found in the blood of human and experimental
cases, and in the tissues and eggs of infected ticks. The
classification of the organism is not yet clear; of its bacillary
form and multiplication by transverse division there can be
no question. The characteristics not common to most bacteria are its coloration with Giemsa's stain, the character of
the reaction excited in tissues and its abundant distribution
within smooth muscle cells of blood vessels. To these differences may be added its probable low specific gravity, as
shown by Ricketts, who found that the virus could not be
completely thrown down by centrifugalization at two thousand
revolutions per minute, during a period of six hours. The
peculiarities in distribution and the staining reaction would
indicate that the organism partakes somewhat of the characteristics of spirochetes. All attempts at cultivation have
thus far failed. It is hoped that this communication will
stimulate others in this endeavor.
Through the invitation of the Montana Department of
Health and State Board of Entomology it is hoped soon to
verify these results upon human cases.
[I wish to express my thanks and appreciation of the services rendered
by Mr. W. W. Chapman of the Third Year class of the Harvard University Medical School, for assistance in maintaining the strain and observations upon the large number of guinea-pigs used.]
I126
WOLBACH.
DESCRIPTION OF PLATE VI.
FIG. i. -Smear preparation of edematous subcutaneous tissue from
scrotum; guinea-pig; Giemsa stain; 2,0oo diameters.
FIG. 2.- Arteriole from skin of guinea-pig; region of sxcrotum;
Loeffler's stain differentiated with dilute acetic acid; 2,000 diameters.
FIG. 3.- Small vein; epididymis; guinea-pig; Loeffler's stain differentiated with dilute acetic acid; 2,000 diameters. Note presence of
organisms throughout circumference of the vessel.
VOL. XXXIV. PLATE VI.
JOURNAL OF MEDICAL RESEARCH.
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Rocky Mountain Spotted Fever.