野口英世著

Journal of Experimental Medicine

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CULTIVATION OF RICKETTSIA-LIKE MICROORGANISMSFROM THE ROCKY MOUNTAIN SPOTTED FEVER
TICK, DERMACENTOR ANDERSONI.
BY I-IIDEYO NOGUCI-II, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
PI~TES 15 TO 18.
(Received for publication, January 7, 1926.)
A large number of microorganisms have been reported in recent years under thename of Rickettsia, or as Ricketlsia-like microorganisms. The majority of theseminute, for the most part intracellular, Gram-negative forms have been found ininsects and arachnids. 1,2 Only in Rocky Mountain spotted fever,8,4 typhus, 5 andheartwater disease 6 have pathogenic Ricketts~ been definitely found in mammaliantissues.
Only a few of the Rickettsice have been isolated in pure culture. NSller ~ reports
successful cultivation of Rickettsia melopkagi, Kuczynski s of R. prowazeki, andSikora g of R. pediculi. By means of tissue culture technique, Wolbach andSchlesinger I° obtained virulent cultures of R. prowazeki and Dermacentroxenus
rickettsi from the tissues of experimentally infected animals.

Since the various Rickatsice thus far described are defined for themost part only by their host species and their morphological features,
it is difficult to decide, in the lack of a comparison under controlled
conditions, whether the forms found in different insects, notwithstanding their morphological similarity, are specific in each instance for thet Cowdry, E. V., J. Exp. Med., 1923, xxxvii, 431.
2 Hertig, M., and Wolbach, S. B., I. Med. Research, 1923-24, xliv, 329.
3 Wolbach, S. B., J. Meal. Research, 1919, xli, 1.
4 Nicholson, F. M., J. Exp. Meal., 1923, xxxvii, 221.
b Wolbach, S. B., Todd, J. L., and Palfrey, F. W., The etiology and pathology
of typhus, Cambridge, 1922.
* Cowclry, E. V., J. Exp. Med., 1925, xlii, 231, 253.
TN/filer, W., Arch. Schiffs- u. Tropen-Hyg., 1917, xxi, 53.
s Kuezynski, M. H., Med. Klin., 1920, xvi, 706, 733, 759.
o Sikora, H., Arch. Schiffs- u. Tropen-Hyg., 1921, xxv, 123.
1o Wolbach, S. B., and Schlesinger, M. J., Y. Med. Research, 1923-24, xliv, 231.
515

516

RICK.ETTSIA-LIKE M'rCROORGANIS~S

insect species, or whether the same form or group of forms infests
several insects. Cultivation on artificial media would undoubtedly
furnish more information with regard to their morphological and biological properties.
The present investigation has to do with the microorganisms of
the spotted fever tick, Dermacentor andersoni, and represents the outcome of an attempt to cultivate the causative microorganism of
spotted fever from infected ticks. A suspension of the viscera of an
infected tick yielded on cultivation in a certain medium n a growthof minute, slightly motile, Gram-negative diplobacillary forms morphologically similar to the forms found in smears of the suspension;and 0.5 cc. of the culture (72 hours old) induced typical spotted fever,after 12 days, when intraperitoneally inoculated into the guinea pig.
The fever lasted 6 days, and the animal showed the characteristic
scrotal lesions of spotted fever. Transfers with blood taken at theheight of fever induced typical spotted fever in other guinea pigs.
Subcultures from the original culture, however, failed to induce either
infection or immunity, as did also the original culture when 2 weeks
old. Other immunological tests proved that the organism in question was in no way related to the virus of spotted fever, which had
survived for a time in the original culture. The strain of spotted fever
recovered from it was carried in guinea pigs for some time. The
organism that had been cultivated resembles Dermacentroxenus rickettsi
morphologically; it grows best at temperatures ranging from 15-26°C.,
and not at all at 37°C.
The incident showed that from the tissues of an infected tick a
non-pathogenic microorganism closely resembling Dermacentroxenus
rickettsi may be cultivated on special media. Through the generosity of Dr. R. R. Parker, of Hamilton, Montana, in keeping me supplied with adequate numbers of ticks, I have been able to carry out asystematic study of a large amount of material, both infective and noninfective.
11To 9 parts tIiss serum water containing various carbohydrates in series wasadded 1 part of 2 per cent nutrient agar. ~/10 I-ICIwas added to give a pI-I range
of approximately 4.5 to 7.4.

HID~YO ~OOUCHZ

517

Material and Mode of Investigation.
A number of ticks collected in the Bitter Root Valley, and termed "drag"ticks because of the method of collection, were fed on guinea pigs infected with astrain of spotted fever virus known as Barlow (which was given to me by Dr. R. R.
Spencer, of Hamilton, Montana, in 1922) for 72 to 96 hours. Those which became
engorged were kept at 12-15°C. during the period before use, which was never lessthan 7 days. Some drag ticks were fed on normal guinea pigs to determine thepresence of natural infection, and in this way three new strains of spotted fever
virus were obtained; these were termed Parker, Sawtooth, and Blodgett. The
three guinea pigs which became infected from the naturally infected ticks wereused for infecting other drag ticks. There were acquired, in this way therefore,
four lots of drag ticks, each lot infected with a different strain of the virus.
M o s t of the ticks fed on n o r m a l guinea pigs did not induce infection,a n d these were regarded as non-infective ticks. T h e infectivity or
non-infectivity of a given tick was determined in n o r m a l guinea pigs
b y feeding a n d b y inoculation of emulsified viscera, or, when theseprocedures g a v e negative results, b y i m m u n i t y tests.
A tick which had become well engorged by feeding 72 to 96 hours was sterilized
as far as possible on the outside with alcohol, ether, and sterile saline solution, andthen cut into halves longitudinally with a sterile knife. One half was put into
Regaud's fixative for sectioning and staining with Giemsa's and Gram-Weigert's
stain. The other half was emulsified in a sterile mortar with 3 cc. of sterile saline
solution. 1 cc. of the emulsion was injected intraperitoneany into a male guinea
pig (450 to 500 gm.), and the remainder inoculated into various media. Smears
were always made with the emulsion and sometimes also with the cut edge of thehalf tick before it was put into the fixative. This latter method often gave thebest results. The smears were stained with Giemsa's and Gram's solutions.
All guinea pigs injected with the emulsions, unless they had typical spotted
fever, were subsequently tested for jmmlmitywith blood from infected guinea
pigs.

Cultural Methods.
I n order to obtain c o m p a r a b l e results, n e a r l y equal n u m b e r s of
infective a n d non-infectlve ticks were studied a t the s a m e t i m e a n d
u n d e r parallel conditions. W h e t h e r or not a given tick was infective
was of course still u n k n o w n a t the time when it was killed for cultivation purposes.

518

RICKETTSIA-LIKE MICROORGANISMS

Several culture media were always used, and~ duplicate series were placed at
26°C. and 37°C. The special media used were: (1) leptospira medium (semisolid)
plus carbohydrate (glucose, saccharose, maltose, mannose, levulose, mannitol,
dextrin, xylose, inulin, salicin, dulcitol, and rbamuose) in a concentration of 0.2
per cent; (2) the same, containing a piece of fresh rabbit kidney; (3) the same as(1), but with an agar concentration of 1 per cent and slanted; (4) Novy and
McNeal's medium containing the same sugars as the foregoing. Plain broth,agar slants, and yolk slants were always employed in addition to the special
media. In the earlier experiments ordinary leptospira medium with and without
fresh tissue was used, but it was soon omitted as less suitable than other media,
notwithstanding the fact that Leishmanias, as well as leptospiras, grow vigorously at 15-26°C. on it, as they do also on media (1), (2), and (4). The quantityof tick suspension used for inoculation of the media was 0.1 cc.
As was to be expected, some of the emulsions contained more than one kind of
microorganism. In such instances the growth obtained on semisolid medium wasplated on the No. 4 medium to separate the organisms.
I~ESULTS.
S e v e r a l varieties of b a c t e r i a h a v e b e e n isolated f r o m emulsionsr e p r e s e n t i n g 75 different ticks. T h e one m o s t f r e q u e n t l y e n c o u n t e r e d
w a s v e r y similar to t h a t isolated in the p r e l i m i n a r y e x p e r i m e n t . T w oo t h e r s often grew on t h e special m e s a , s o m e t i m e s t o g e t h e r . Allt h r e e of t h e varieties c u l t i v a t e d h a v e certain m o r p h o l o g i c a l f e a t u r e s
w h i c h s i m u l a t e t h e p l e o m o r p h i c p h a s e s of Dermacentroxenus rickettsi
in s p o t t e d f e v e r ticks, as described b y p r e v i o u s i n v e s t i g a t o r s . F o rt h e s a k e of convenience, I shall designate t h e first t y p e Bacillus
rickettsiformis, t h e second B. pseudoxerosis, a n d t h e t h i r d B.

equidistans.
Bacillus rickettsiformis, N. Sp.
Growth.--Initial cultures were obtained only on the special media, and at atemperature of 26°C., as minute, yellowish gray, semitransparent, shining, oval orround, slightly raised colonies near the condensation water. Consistency somewhat mucoid; after several days the colonies show a tendency to coalesce. The
condensation water is slightly turbid and contains a grayish, rather sticky sediment. In a few instances a grayish surface growth was obtained on leptospiramedium. Old strains grow well on broth, which shows slight opalescence, withsurface pellicle and deposit, and on ordinary agar, on which some strains form alight yellowish pigment after several days. No growth is obtained under anaerobic
conditions. One strain by gradual adaptation was made to grow at 37°C. Gelatin is not liquefied.

HIDr.YO NOGVCm

519

Morphology.--In young cultures the organisms are fairly uniform in size,
measuring 0.3~,in width and 0.75~,in length; some of them are moderately motile.
Single organisms are lanceolate, fusiform, or rod-shaped, and measure 0.25 to
0.35~ in width at the widest portion and 0.5 to 1~,in length, the longest about 1.4~.
Diplobacillary forms consist of two lanceolate forms joined end to end and measure about 2~t in length. The long rods do not occur in diploid form. In old cultures there is considerable pleomorphism, some individuals becoming oval, fusiform, or filamentous; the filamentous forms may show unevenness in width
throughout the length of the body; some, especially those which settle down tothe bottom of the fluid medium, have swollen ends. Vigorously growing individuals take a better stain than old ones. The organism is Gram-negative; it does
not take Loeffier's methylene blue well. Staining with Giemsa's solution showsa light reddish violet body, of bipolar or diplobaciUary shape, the middle portion
being less distinctly stained. In bipolar elements the ends are abruptly pointed
and show no stricture at the middle portion of the organism. Under certain conditions almost coccoid shapes may be assumed.
Strain Variations.--Variations in size exist among different strains, some being
decidedly coarser or finer than the average type. In old surface colonies of somestrains a light lemon-yellow color may appear.
Photomicrographs of the organisms found in smears and sectionsof infective and non-infective ticks, and of cultures of B. rickettsiformis, are shown for comparison (Figs. 1 to 23). Figs. 1 and 2 aredark-field views of one of the richest smears I have ever seen from
non-infective ticks. T h e organisms are diploid and appear in paired
refractile oval dots close together. In the center of Fig. 1 is a large
round mass of organisms which look as if t h e y were packed in a sac.
This appearance is characteristic of rich emulsions.
When one compares carefully the forms found in the ticks and thoseassumed by B. rickettsiformis under various cultural conditions, one
is t e m p t e d to regard t h e m as identical. T h e causative agent ofspotted fever, Dermacentroxenus rickettsi, is indistinguishable morphologically from the forms found in infective ticks, which again are similar to those found in non-infective ticks and those assumed b y B.
rickettsiformis; the only points of differentiation in the case of Dermacentroxenus rickettsi are its pathogenicity for certain mammalians andthe fact t h a t it is uncultivable b y the methods employed in this investigation. Since ticks in general harbor in their tissues so called Rickettsi¢, the non-pathogenic tissue parasites in some of the wood ticks m a ylikewise be regarded as Rickettsia, according to the present d a y usage

520

RICKETTSIA-LIKE~flCROORGANIS]~fS

of the term, which is based on morphological characteristics. Should
similar organisms be found in ticks collected in the eastern parts of
the United States, we shall have an explanation of the presence, in
non-infective ticks caught in spotted fever regions, of these forms
which are morphologically so confusing when studied in connection
with the spotted fever organism.
The foregoing morphological description applies to tick smears
equally as well as to cultures of B. rickettsiformis. Some very minute(0.25 by 0.5/~) and some rather large (0.4 b y lv) individuals occur insmears as well as in cultures, as do also coccoid forms (0.35 b y 0.4/~).
In tick sections (salivary glands, etc.) single lanceolate individuals(0.25 to 0.35# to 0.5 by 1/~) predominate; in some sections thinner
forms are found, owing to different conditions of fixation and differentiation (Figs. 14, 19, 20). This appearance is also observed incultures (Figs. 21, 22). Some coarser individuals are found in muscle(Fig. 18).
The organism of Rocky Mountain spotted fever in mammaliantissues presents striking resemblances to B. rickettsiformis, notwithstanding the fact that the latter is not related to it in any way. Smears
show somewhat coarser organisms (Figs. 24 to 26) than sections (Figs.
27 to 29), owing to flattening of the former and greater decolorizationof the latter in differentiation. Similar forms are seen in the smearsof cultures of B. rickettsiformis (Figs. 30 to 34). Rather coarser individuals but with similar morphological characteristics are seen in
Fig. 35.

Bacillus pseudoxerosis, Y. Sp.
Growtk.--Initial growth was obtained on the special media (slanted and semisolid) at 26°C. The colonies are light yellowish gray, shining, mucoid, and confluent. Subcultures grow fairly well on plain agar and broth, producing in thelatter slight turbidity, with surface scum and stringy sediment. Optimum temperature for growth, 26°C. No growth under anaerobic conditions. Gelatin
not liquefied.
Morphology.--Non-motile, slender, pleomorphic bacilli, measuring, in young
surface cultures, 0.3 to 0.5/~ in width and 0.4 to 2~ in length (Fig. 36).
There is some resemblance to B. xerosis in arrangement and appearance. In
somewhat older cultures groups of short rods are mingled with longer, often considerably thicker, rather curved individuals, 0.5 to 0.55u by 3 to 4u (Figs. 37,
38). The organism is somewhat resistant to Gram's stain. Bifurcated forms
are seen in some cultures (Fig. 38).

Hm~YO NOGVCm

521

Bacillus equidistans, N. Sp.
Growth.--Initial growth was obtained on the special slanted and semisolid
media at 26°C. Light grayish white, somewhat fluorescent, round or oval, shiningcolonies on the surface of slants, tending to become confluent within several days.
Growth of later subcultures obtained on plain broth and agar slants. The broth
becomes moderately turbid, and after several days there is a heavysticky scum andsediment. Colonies on slants are at first delicate but in a few days become heavier,mucoid, and have a light, brownish yellow tint. The organism grows best at
15-26°C.; 37°C. is less favorable for growth. No growth occurs under anaerobic
conditions. Gelatin is not liquefied.
Morphology.--The most characteristic feature of this organism is its appearanceof uniform dispersion, as if the individuals were purposely placed equidistant fromone another. No motility has been observed. In young surface cultures there
are many single individuals measuring 0.25 to 0.3~ by 0.4 to 0.8v (Fig. 39).
Diploid forms are sometimes predominant (Fig. 40), and there is an appearance ofa narrow bridge between the two cells. In some instances minute coccoid forms,
not unlike Bacterium tularense, may be assumed. This organism is negative by
Gram's stain and stains reddish violet with Giemsa's solution. It appears to havea capsule, as indicated by a clear unstained or lightly stained zone several times
wider than the cell itself. In older cultures are found irregularly curved filamentous forms, with a tendency to branch (Fig. 41).
T h e feces of ticks yielded the same three types of organisms on
cultivation, B. rickettsiformis being most frequently present. I t m a y
be mentioned t h a t the injections of suspensions of feces from t w e n t yticks which were infective (as shown b y production of spotted feverb y feeding on guinea pigs) produced some local infiltration and rise oft e m p e r a t u r e within 48 hours, b u t no spotted fever symptoms nor
a n y i m m u n i t y to the disease. This experiment merely shows theinfrequency of spotted fever organisms in the feces of infected ticks
and does not exclude the possibility of their occasional presence.
T h e results of fermentation tests with the three varieties of microorganisms cultivated are recorded in T a b l e I. T h e medium used was
I-Iiss's litmus serum water, and the cultures were k e p t 7 days at 26°C.
T h e strains of B. rickettsiformis used in this test only slightly
a t t a c k e d dextrose, galactose, levulose, and lactose. In some otherexperiments, however, strains were encountered which caused slight
acidity with dextrose, maltose, xylose, and dextrin. B. pseudoxerosis
affected the m a j o r i t y of carbohydrates employed b u t never sufficiently
to cause a n y coagulation. B. equidistans fermented dextrose sufl~-

522

R I C K E T T S I A - L I K E MICROORGANISMS

ciently to produce marked acidity and coagulation without evolution ofgas. Other carbohydrates were either very slightly or not at all
fermented. In this respect the organism, though perhaps belonging
to the group of B. mucosus capsulatus, appears to differ from any of
the group so far known.
TABLE I .

Fermentation Reactions with the Microorganisms Cultivatedfrom Ticks.
B. ricke~formls.

B1 pseudo~o~is.

B. equldistavs.

7 days.

30days.

7 days.

3O
days.

Dextrose . . . .

Trace acid.

+

Distinctly acid.

+++

Saccharose...
Galactose . . . .
Maltose.

Trace acid.

Levulose. . . .

Trace acid.

......

Mannose..
Mannitol..
Dulcitol...
Rai~ose.
Rhamnose . . . .
Dextrin..

Inulin. . . . . . .
Salicin . . . . . . . .

3O
days.

Coagulation; + + +acid; no gas.

+++
Slightly acid.

Xylose.
Lactose7 days.

Trace acid.

..~

m

lg

lc

gg

ll

IC

ll

+++
+++
+
++
+++
+++

++

....
....

++-~
++

Decolorized;
no acid.

--

Slightlyacid.

++

m

-4-

-t-

Trace acid.
gg

ll

**

a*

--

--

+++
+++
+++
+

Markedly+ + -~
acid; no gas.
Trace acid.

Frequency and Distribution of the Various Organisms among Wood
Ticks.
Most of the tick emulsions were sterile as tested with plain media(agar and broth) and with special media at 37°C., but the majority
yielded some colonies on the spedM media after several days incubation at 26°C. Subsequent purification by plating showed that most of
the ticks contained B. rickettsiformis, some B. pseudoxerosis or B.
equidistans in addition, while a few yielded only one of the latter. In
Tables I I to VI are recorded the data regarding the findings in thecase of each tick, including the results of biting tests, injection of

523

HIDEYO N O G U C H I

e m u l s i o n of v i s c e r a , a n d s u b s e q u e n t t e s t s for specific i m m u n i t y a g a i n s t
s p o t t e d f e v e r , t h e p r e s e n c e o r a b s e n c e of m i c r o o r g a n i s m s in s m e a r s a n ds e c t i o n s , a n d f i n a l l y t h e v a r i e t i e s of b a c t e r i a c u l t i v a t e d .
T A B L E 1-[.

Drag Ticks Which Did Not Induce Spotted Fever When Tested on Normal Guinea
Pigs 12 Days Previously.
Results ofTick No.

Inoculation of Immunity Smears.
emulsion,
test.

Feeding.
1

.

2

--

3

.

.

4

.

.

5

--

6

.

7

--

8

.

9

.

10

.

[

.

.

.

--

--

--

.

.

B. rickett$iformis.

.

+

.

.

+

.

-.

.

.

<+

.

B. rickettsiformis.

.

.

--

.

+

.

.

.

.

<+

.

--

Bacteria isolated in culture.
Sections.

.

+++

.

.

.

B. rickettsiformis.
.

B. rCckettsiformis; B. pseu-<+

doxerosis.
.

.

.

.

.

11

.

.

.

.

.

.

12

.

.

.

.

.

.

13

.

.

.

.

14

.

.

.

15

--

I

16
17
18
19
20

-.
--.

--

21

.

22
23
24
25

.
.
.
--

l

--

.

.
--.
.

.
.
.
--

+ +

B"

pseudoxerosis.

+

++

"

"

.

.
+++
++++
++
++++
.
.
.
?

.
.
.
--

.

+

.

.
.
.

.

.

.

---

.
.

.

.

.
.
.
+

B. pseudoxerosis.
" rickettsiformis.

++

B. rickettsiformSs.

.
.
.
++++

B. rickettsiformis.

A n a l y s i s o f R e s u l t s S h o w n i n T a b l e s I I to V I .

Of 25 t i c k s f e d p r e v i o u s l y on n o r m a l g u i n e a pigs, n o n e c o n f e r r e d
e i t h e r i n f e c t i o n (as t e s t e d b y b i t i n g e x p e r i m e n t s o r i n o c u l a t i o n ofe m u l s i f i e d v i s c e r a ) o r i m m u n i t y ; t h a t is, t h e y w e r e n o n - i n f e c t i v e u n d e r
the present experimental conditions.
15 of t h e s e y i e l d e d n o c u l t u r e ,524

RICKETTSIA-LIKE

MICROORGANISMS

5 yielded strains of B . rickettsiformis, 3 B . pseudoxerosis, and 2 B.
rickettsiformis and B . pseudoxerosis.
Of 49 ticks fed previously on spotted fever guinea pigs, 14 conferredneither infection nor immunity; of these 2 yielded no cultures, 5yielded B . rickettsiformis, 2 B . pseudoxerosis, 2 both of these organisms,
1 B . equidistans, 1 B . pseudoxerosis and B . equidislans, and 1 an undetermined Gram-negative bacillus.
TABLE III.

Drag Ticks Which Became Engorged When Fed on Spotted Fever Guinea Pigs
(Barlow Strain) 12 Days Previously.
Results ofTid ~'o.
Feeding.

+

I

Inoculation of I~tesU~i'ty Smears.
emulsion.

+<+

Bacteria isolated in culture.
Sections.

+

+
+
+
+

+
+
+
+

<+
<+
+
+

+
+
++
+

+
+
+
+

+
+
+
+

+
+
~~-

++
++
+
+

B. rickettsiformi,~.

,,

,,

-

_

_

+

+

+

+

++

+++

B. rlckettdformis.
. . . .
. . . .
. . . .
",,
. . . .

+

-

+

<+

++++

,,

+

+

<+

<+

"

+

+
+

++
+++

++ +
+++

+

++++

+++

- -

--

+

+

+++

+++

.

"
B. puudoxerosis.
B. rickettsiform~s.
" pseudoxerosis.
" rickatsiformis.
. . . .

Of the remaining 35 ticks, 16 (Group 1) caused typical spotted fever,both by biting and inoculation of emulsions, 13 (Group 2) failed to
infect by biting, but the inoculation of emulsions conferred infection,
4 (Group 3) caused slight fever which proved by subsequent immunity
test to have been due to spotted fever infection, and 2 (Group 4)
were negative, but the animals which received the emulsified viscera
proved to be immune to spotted fever; that is, all of these 35 ticks

nmw¢o NOG~Cm

525

carried the virus of spotted fever, 16 of them in highly virulent form.
10 of the ticks of Group 1 yielded B. rickettsiformis in culture, 2 B.
rickettsiformis and B. pseudoxerosis, 1 B. equidistans, and 3 no growth.
Of Group 2, 1 was sterile, and 12 yielded cultures, 4 B. rickettsiformis,
2 B. pseudoxerosis, 2 both, 3 B. rickatsiformis and B. equidistans, and
1 B. equidistans. Of Group 3, 1 was negative and 3 positive, 1 yielding B. rickettsiformis, 2 B. pseudoxerosis. One tick of Group 4 yieldedrickettsiformis the other rickettsiformis and pseudoxerosis.
TABLE

IV.

Drag Ticks Which Became Engorged When Fed on Spotted Fever Guinea Pigs (Parker
Strain) 12 Days Previously.
Results ofTick to.
Feeding.

Inocula- Immunity
tion oftest.
emulsion.

Bacteria isolated in culture.
S

m

~

s

.

++

S~dom.

+++

B. rickatsiformls;B. ps~doxerosis.

B

m

m

+

+

+

++++

+

+
+
<+

+++
++
<+

++++

+++

+
+

B. rickettsiformis; B. pseudoxerosis.
B. rickcffsiformis.

m

+

+

+

+
+

+
+

++++ ++++<++++

B. ricketts~ormis; B. pseudoxerosis.
B. rickegs'iformis.
""
B. equidiaan~.

The percentage of non-infective ticks which yielded cultures was
40 per cent, of infective ticks 85 per cent. B. rickettsiformis occurredalone in 27 of the 52 ticks from which cultures were obtained (51 percent), and in association with one of the organisms in 11, the total
incidence of this organism being 73 per cent. B. pseudoxerosis
occurred alone in 9 instances, and in association with one of the others
in 9, the total incidence being 33 per cent, while B. equidistans occurredalone in 3 and with one of the others in 4 instances, the total incidence
being 13 per cent.

526

RICKETTSIA-LIKE

MICROORGANISMS

TABLE

V.

Drag Ticks Which Became Engorged When Fed on Spotted Fever Guinea Pigs
(Sawtooth Strain) 12 Days Previously.
Results ofTick No.

Feedi~.

Bacteria isolated in culture.

Inoculation ofemulsion.

Immunity
test.

Smears.

Sections.

+

+

+++
+

+

+

+++
+
<+
<+

m

<+

+

+
+

+
Fever.

+

+

+

10
li

+

--

Fever.

+

+

M

++

++++

+

+

++

+++++

+++

+++++

B. pseudoxerosis.
" ricketts~ormis.
""
B. pseudoxerosis.
" equidistans.
" rickettsiform';s; B. pseudoxerosis.
" pseudoxerosis.
""
B . equidistans.
B. rickeCtsiformis; B. equi.
distans.
B. rickettsiformis; B. equidistans.
B. rickettsiformis.

T A B Lt~- V I .

Drag Ticks Which Became Engorged When Fed on Spotted Fever Guinea Pigs
(Blodgett Strain) 12 Days Previously.
Results ofT i c k No.

Bacteria isolated in culture.

Inoculation ofemulsion,

ImmunitytcsL

Smears.

Sections.

_

_

<<+

+

---

N
~

-~-

m
m

_

_

<+

+
+
+++

_

_

<+

Fever.
"
+
+

+
+
+
+

?
+
++
-"

Feeding.

5

- 4 -

6
7
8

B

9

m

10

11

+

+++
+
+
+
+
++
+

B. rickegtsiformis; B. pseudoxerosis.
B. pseudoxeros~.
" ricketgsiformis.
Minute, motile, hemolytic,
Gram-negative bacillus.
B. equidistana.
" rickettsiformis.
" pseudoxerosis.
B. equidistans.

Emv.Yo ~oGvcm

527

Comparison of the cultural findings with the microscopic examination of the smears and sections shows that 17 ticks which conferredneither spotted fever nor immunity to the infection were negative both
microscopically and by culture. All those ticks which yielded culturesof one organism or the other were positive also by microscopic examination, containing minute Gram-negative organisms in varying numbers in smears and sections, in some instances in enormous numbers.
On the other hand, in 5 instances in which the ticks were definitely
infective, the smears and sections showed the familiar Gram-negative
organisms, yet no cultures were obtained from the emulsions. These
forms may have been the true spotted fever organism, Dermacentroxenus rickettsi, and, like similar organisms in tissues from infectedanimals, could not be cultivated by the methods employed in thepresent investigation. The most striking point, however, is the morphological and tinctorial resemblance between the non-pathogenicorganisms and the spotted fever parasites.
The source of the non-pathogenic microorganisms is unknown. I
have never encountered any of them in mammalian bacterial flora,
and they do not grow at body temperature. The optimum temperature of 12-26°C. or lower dearly indicates their adaptation to insects.
For comparison, 5 drag ticks which had never been fed on guinea pigs
were studied culturally. One of these contained B. rickettsiformis,
the others were sterile. The result shows, however, that B. rickettsiformis does occur under natural conditions in the wood tick. The
high incidence of these non-pathogenic parasites in the wood ticks used
for the present study may not indicate their presence in wood ticks ingeneral, for ticks collected in different localities and at different
periods may not yield the same results.

Pathogenicity and Immunological Properties of the Bacterial Strains.
Guinea pigs, rabbits, and monkeys (Macacus rhesus) were inoculatedwith young cultures of B. rickettsiformis, B. pseudoxerosis, and B.
equidistans grown on serum-containing media. In no instance wereany lesions of spotted fever induced. There were a number of
instances in which a continuous high temperature developed andlasted 2 to 5 days, but the spleen was usually normal, and most of the528

RICKETTSIA-LIKE

MICROORGANISMS

animals recovered within a week. They were later tested for theirsusceptibility to the spotted fever virus, but in only two instancesamong 69 animals was any immunity present. In the two instancesin which the animals became resistant to spotted fever, no significancewas attached to the observation, because other animals which hadreceived the same culture developed typical spotted fever.
Several rabbits were immunized with B. rickettsiformis, B.
pseudoxerosis, and B. equidistans, respectively, by intravenous injection of cultures, and the immune serums were tested for neutralizing
properties for the spotted fever virus. The results were negative.
The action of spotted fever immune serums from hyperimmunized
rabbits TM and from recovered human beings TM was tested in vitro
upon each of the three organisms, but no specific agglutination orcomplement fixation was demonstrated. There was a tendency to
the formation of clumps in some instances with human or rabbit
serums (including normal ones) upon 24 to 48 hours standing at room
temperature, owing to slow multiplication of the organisms.
The three organisms were also tested for agglutination and complement fixation with anti-tularense immune serum kindly furnished by
Dr. Edward Francis, but the results were negative.
With regard to the use of tick emulsions as antigens in specificcomplement fixation tests with immune serums prepared in rabbits
b y repeated injections of virulent blood from infected guinea pigs, it
was found that ticks fed on guinea pigs 7 days previous to the test stillcontain enough guinea pig blood to give strong fixation; of 20 ticks fed
40 days previously, fixation was still complete with the emulsions of 3
(females). The immune serum used for this work was of a titre suchthat 0.00001 cc. of guinea pig serum still completely fixed complement.
To avoid this difficulty, human and guinea pig convalescent serums
were tested with infective and non-infective tick emulsions. The
results were uniformly negative, except in the case of two human(one normal, one convalescent) and two rabbit (one normal, one
immune) serums, which fixed complement.
x~Noguchi, H., J. Exp. Med., 1923, xxxvii, 383.
18The specimensof serum from persons recovered from Rocky Mountain spotted
fever were sent me through the kindness of Dr. W. F. CogsweUand Dr. John X.
Newman, of the State Board of Health of Montana.

amxYo ~OGVCM

529

SUMMARY.

A systematic study of 74 ticks, the infectivity or non-infectivityof which was determined by biting experiments, inoculation of emulsions, and specific immunity tests, showed the presence in someinstances of several types of microorganisms morphologicallyresembling the inciting microorganism of spotted fever. The mostfrequently isolated was B. rickettsiformis, n. sp., those less commonly
encountered were B. pseudoxerosis, n. sp., and B. equidistans, n. sp.
These organisms are non-pathogenic for the guinea pig, rabbit, andMacacus rhesus. In morphological features they resemble the forms
found in smears and sections of the ticks, yet their presence had no
relation to infectivity. Immunologically they are not related to thespotted fever virus. All three are pleomorphic under cultural conditions, and the question arises whether or not the minute non-pathogenic Rickettsia forms and the somewhat coarser symbionts found in
Dermacentor andersoni are morphological variations due to variations
in nutrition, oxygen tension, tissue reactions, etc., in the different tissues and cells in which the organisms are embedded. At all events,
the differentiation of the non-pathogenic Rickettsia-like organisms from
Dermacentroxenus rickettsi is extremely difificult. 1. In definitely infective ticks of the present study intranuclear forms were not constant.
A point of special interest is that these non-pathogenic microorganisms from ticks grow best at room temperature, in this respect
resembling culturally certain flagellates inhabiting the alimentarytract of insects. 16 The difficulty of obtaining initial growth on artificial media and the gradual adaptation to less specialized media areother notable characteristics of these organisms. The possibility thatB. rickettsiJormis is a non-pathogenic phase of the spotted feverorganism, comparable with the avirulent flagellate culture forms of
14Dr. E. V. Cowdry has been kind enough to look through the preparations
described in this report without being informed whether they came from infective
or non-infective ticks, in order that he might give an objective opinion concerningthem. The sections of non-infective ticks contained forms which Dr. Cowdry
regarded as Dermacentroxenus rickettsi; that is, forms indistinguishable from thosefound in sections of infected ticks. The organisms occurred in the salivary glands
in both infective and non-infective ticks.
15Experiments to be reported upon later.

530

RICKETTSIA-LIKE MICROOllGANIS~S

Ldshmanias, seems r e m o t e in view of the n e g a t i v e i m m u n o l o g i c a lfindings.
H e r e d i t a r y transmission of B. rickettsiformis is d e a r l y i n d i c a t e d b y
its presence in large n u m b e r s in ovaries a n d egg cells, a c h a r a c t e r istic also of the s p o t t e d fever o r g a n i s m ~6 a n d of o t h e r i n s e c t - b o r n e

Rickettsice.t,
EXPLANATION OF PLATES.
PLATE 15.

FIGS. 1 and 2. Dark-field views of an impression film preparation from a noninfective tick. Film fixed in methyl alcohol, stained with Giemsa's solution, andphotographed by dark-field illumination. Note the large unresolved mass oforganisms in the left half of Fig. 1. X 900.
Fio. 3. Dark-field view of B. rickettsiformis cultivated for 6 days at 26°C. on
Hiss serum water containing 1 per cent salicin. Note several agglomerates of theorganism from which some individuals have become partly dissociated. × 1000.
FIG. 4. Same film preparation as Figs. 1 and 2, photographed in the ordinary
way. X 1000.
FIO. 5. Smear preparation of an infective tick emulsion, fixed with bufferedformalin. Gram (negative) counterstained with saturated alcoholic solution offuchsin. X 1000.
Fro. 6. Smear preparation of an infective tick emulsion, stained in the same
way as Fig. 5. X 1000.
FIG. 7. A cell from the hypoderm of an infected tick. Zenker's fixation followed by Giemsa's stain. Note the numerous rod-shaped organisms filling thecytoplasm of the cell. × 1000.
FIG. 8. Film preparation of the brain of an infected tick. Giemsa's stain.
X 1000.
F1G. 9. Culture of B. rickettsiformis grown on ascites agar slant for 6 days at
26°C. Giemsa's stain. X 1000. Note the striking resemblance to the forms
shown in Fig. 4.
Fro. 10. Culture of B. rickettsiformis. Blood slant 8 days old (26°C.).
Giemsa's stain. × 1000.
FIG. 11. A young culture of B. rickettsiformis on blood sugar slant 48 hours old(26°C.). Giemsa's stain. × 1000.
FIG. 12. B. rickettsiformis on NSller's medium (slant) grown 48 hours at 26°C.
Giemsa's stain. X 1000. The forms shown in Figs. 11 and 12 bear a remarkable
resemblance to those described by Wolbach (Fig. 34, Plate VII, J. Med. Research,
1919, xli, 1-197).
16Ricketts, H. T., J. Am. Med. Assn., 1907, xlix, 1278.

m~zYo Nooucm

531

P z A ~ 16.
Magnification × 1000. Giemsa's stain.
FIGs. 13 and 14. Rickalsiaolike organisms in the ceils of the salivary gland of
a non-infective tick. Regaud's fixation.
FIGS. 15 and 16. Similar organisms in the salivary ducts of a non-infective
tick. Regaud's fixation.
FIG. 17. A cell of the hypoderm of a non-infective tick. Regaud's fixation.
FIo. 18. Similar organisms in the muscle fibres of a non-infective tick. Regaud's fixation.
FIG. 19. Rickettsia-like organisms in the epithelial wall of the rectal sac of an
infective tick.
FIG. 20. Similar organisms in the brain of an infective tick (Dermacentroxenus
rickatsi?). While these forms are decidedly more minute than some of theorganisms found in sections of non-infective ticks, yet the minute forms shown in
Fig. 14 (non-infective tick) are not morphologically and tinctorially distinguishable from those in the sections of infective ticks.
FIo. 21. B. rickatsiformis grown on slant agar for 8 days at 26°C.
FIo. 22. Same organism grown on serum agar slant for 8 days at 26°C.
Fxo. 23. Same organism grown on Carrel's tissue culture medium for 3 days at
37°C.
All the forms found in the sections of ticks are to be observed aLsoin these cultures.
PLATS 17.
Magnification × 1000. Giemsa's stain (except Fig. 31).
Fxo. 24. Impression smear of the spleen of a guinea pig experimentally infectedwith the spotted fever virus. Buffered formalin fixation.
FxG. 25. Film preparation of teased lesions of tunica of a guinea pig infectedwith spotted fever. Buffered formalin fixation.
FIG. 26. Impression smear of the ear lesion of a guinea pig infected with spotted
fever, showing an unusually large clump of spotted fever organisms in characteristic arrangement.
FxGs. 27 to 29. Spotted fever organisms in the endothelial cells of the small
blood vessels at the sites of spotted fever lesions in guinea pigs. Regaud's fixation.
FIG. 30. B. rickatsiformis grown on ascites agar slant for 5 days at 37°C. (after
gradual adaptation to this temperature).
Flos. 31 and 32. Same organism grown 4 days at 37°C. on blood sugar slant.
Fig. 31 Gram's stain (negative) counterstained with fuchsin; Fig. 32 Giemsa's
stain.
FIO. 33. Same organism grown in serum broth at 37°C. for 6 days.
FIo. 34. Same organism grown on ascites broth at 37°C. for 3 days.
Fxo. 35. Same organism grown on tissue broth at 37°C. for 8 days.

532

RICKETTSIA-LIKE ~[ICROORGANISMS

These photographs show the close resemblance of the forms found in cul l r ~ of

13. rickettsiformis grown under different conditions to the spotted fever orga ism inmammalian tissues, although B. rickettsiformis is not pathogenicaUy r immunologically related to the causative organism of spotted fever.
PLATE 18.
Magnification X 1000. Giemsa's stain.
FIG. 36. B. pseudoxerosis grown on agar slant for 3 days at 26°C.
FIGs. 37 and 38. Same organism grown in broth for 5 days at 26°C.
F i t s . 39 and 40. B. equidistans grown on agar slant for 3 days at 260C.
Flc. 41. Same organism grown on broth for 5 days at 26°C.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X L I I I .

PLATE 15.

(Noguchi: Ri~kellsia-like microorganisms.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. X L I I I .

PLATE 16.

(Noguchi: Rickettsia-like microorganisms.)

THE JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X L I I I .

PLATE 17.

(Noguchi: Rickettsia-like microorganisms.)

T H E JOURNAL OF E X P E R I M E N T A L M E D I C I N E VOL. X L I I I .

PLATE 18.

(Noguchi: Rickettsia-like m~croorganisms.)