THE JOURNAL OF DIFECTIOUS DISEASES. VOL. 137, NO.2.

FEBRUARY 1978
© 1978 by the University of Chicago. All rights reserved. 0022·1899/78/3702·0011$00.75

Cyclic Thrombocytopenia Induced by a Rickettsia-Like Agent

in Dogs

John W. Harvey, Charles F. Simpson, From the College of Veterinary Medicine, University of
and Jack M. Gaskin Florida, Gainesville, Florida

Hematologic manifestations and the ultrastructure of a platelet-specific microorga-

nism isolated from a dog in Florida were studied. The agent was readily transmitted
experimentally to adult dogs by intravenous inoculation with infected blood. Para-
siternias and concomitant thrombocytopenias were cyclic in that both recurred within
relatively constant periods of one to two weeks following experimental infections.
Hemorrhage was not a manifestation of the disease even though thrombocytopenias
were severe. Microorganisms were visualized by light and electron microscopy. They
were observed only in platelets and were composed of single or multiple subunits

Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015

(morula forms). The microorganisms were ultrastructurally very similar to those
reported in Ehrlichia canis infections of dogs and Anaplasma marginale infections of
cattle. Microorganisms were surrounded by single membranes which more or less
conformed to the external surfaces of subunits that were surrounded by double mem-
branes. From electron microscopic studies, it is suggested that these organisms be
classified in the order Rickettsiales.

Thrombocytopenia has been reported in studies Materials and Methods

of a variety of diseases of people and animals, but
Animals. Healthy adult dogs of mixed breed-
the presence of microorganisms within platelets
ing, weighing 16-20 kg, were used. Dogs were
has only occasionally been reported [1-3]. Incor-
dewormed and dipped for internal and external
poration of a microorganism into platelets may
parasites and quarantined for at least three
result from the weak phagocytic capability of
weeks before inoculation. Animals were housed
these cells [1]. Although some viruses infect mega-
in separate runs and were given commercial dry
karyocytes and platelets [2, 4, 5], these viruses also
food and water ad lib. Dogs no. 1 and 3 were male,
infect other cell types. Little evidence of the
and no. 2 and 4 were female. Dog no. 1 had been
specific infection of platelets by microorganisms
splenectomized as a puppy. Dog no. 4 was sple-
is available. An epicellular parasitic species in
nectomized three weeks before inoculation. Dogs
the genus Eperythrozoon (or Haemobartonella)
no. 2 and 3 had intact spleens.
which appears to be platelet-specific has been
Microorganism. The agent used in this study
described in cattle [6].
was originally recovered from a naturally in-
The present study describes the hematologic
fected one-year-old female Keeshond dog in which
manifestations and ultrastructure of a Rickettsia-
small inclusions were noted in 17% of the plate-
like, platelet-specific organism in dogs. These
lets during a routine hematologic examination.
organisms occur and replicate within platelets
The platelet count at that time was 3.3 X 104/JLl,
of infected dogs.
the packed cell volume was 32%, and the total
leukocyte count was 9.5 X UP/ JLl. Clot retraction
Received for publication June 20, 1977, and in revised
was minimal after 24 hr at room temperature
form September 7,1977.
This paper represents no. 605 in the Florida Agricultural (about 24 C). The erythrocyte indices, reticu-
Experiment Stations Journal Series. locyte count, and differential leukocyte count
We thank Jane Boone, 1- W. Carlisle, and Glenda Hall for were all within normal ranges. A volume of 2 ml
technical assistance, and Dr. M. Ristic for performing in- of blood from this dog was injected iv into dog
direct fluorescent antibody tests for Ehrlichia canis.
no. 1. Blood from dog no. 1 during the first para-
Please address requests for reprints to Dr. John W. Har-
vey, College of Veterinary Medicine, Box J-125, J. Hillis sitemic episode was mixed with equal volumes of
Miller Health Center, University of Florida, Gainesville, 15% glycerol in a phosphate-buffered 0.9% NaCI
Florida 32610. solution (pH 7.2) [7]. Aliquots (l-ml) were froz-

182
 Infectious Cyclic Thrombocytopenia 183

en and stored in ampules in liquid nitrogen.
Ampules from this sampling were thawed and
used for subsequent iv inoculations of the three
additional dogs.
Clinical and hematologic evaluations. Each
day dogs were examined for clinical signs and
bled for hematologic evaluation, and tempera-
tures were recorded. Venous blood samples were
collected and stored in 2-ml sealed vacuum glass
tubes (Vacutainer,® Becton-Dickinson Corp.,
Rutherford, N.J.) with EDTA as an anticoagu-
lant. Cover-slip blood films were examined for
organisms with both Giemsa and new methylene
dogs with high percentages of platelet inclusions
were collected, mixed with 3.8% sodium citrate
as anticoagulant, and centrifuged at 500 g for 3
min at 25 C. Platelet-rich plasma was removed,
and platelets were pelleted by centrifugation at
1,000 g for 3 min at 25 C. Plasma was removed,
and pellets were fixed in 2.5% glutaraldehyde in
Sorenson's buffer (pH 7.2) for 2 hr, washed with
the same buffer, fixed in 1.0% OS04 for 1 hr [9,
10], and embedded in plastic (Aldrite 502; Ciba
Products Company, Fair Lawn, N.J.). Thin sec-
tions of platelets 011 grids were stained with
uranyl acetate and lead citrate or uranyl acetate

Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015

blue stains [8]. Routine hematologic determina-
tions were performed with an electronic cell
counter (ZBI-6 System; Coulter Electronics, Hia-
leah, Fla.). Platelet counts were performed with
a hemacytometer chamber and prepackaged cell-
counting diluent (Unopette; Becton-Dickinson
Corp.). Protein concentrations in plasma were
determined by refractometry (American Optical,
Buffalo, N.Y.).
Electron microscopy. Samples of blood from
alone [11] prior to examination by electron mi-
croscopy (EM 200 electron microscope; Philips
Electronics Instruments, Mount Vernon, N.Y.).
Postmortem examinations. All experimental
dogs were sacrificed and necropsied. Brain, liver,
urinary bladder, bone marrow, spleen, mesenter-
ic lymph node, kidney, lung, intestinal tract,
testes, and heart from dogs no. 2, 3, and 4 were
fixed in 10% neutral formalin, embedded in
paraffin, and cut in 5-p.m sections. Sections were

~
~
(J)
I-
w
...J
~ w
(J)
l-
W
...J
W
!q
...J
"1
30

13
I
!q
...J
Q.

c
w
N
15

10

Q.
i=
c
W
N 10
~
a:
i= ~ 0
iii
«
a: 3
~ 10

..
0
8

4
~
""
~ (J)
6

~ I-
w
...J
(J) 4
W
I-
W Z !q
...J ...J
W Q. Z
!q
...J
Q.
0

i i i i I i I i
80 -13 0 13 30 43 60 75

DAYS DAYS

Figure 1. Percentage of parasitized platelets and platelet counts of a nonsplenectomized dog (left) and a splenec-
tomized dog (right) after iv inoculation with blood infected with a platelet-specific parasite. Days are numbered
from day of inoculation.
184 Harvey, Simpson, and Gaskin

stained with hematoxylin and eosin and Giemsa

stains and were examined by light microscopy.

Results

Hematologic effects. Each dog became in-

fected after inoculation. The prepatent period
varied from seven to 12 days. In each case, the
maximal percentage of parasitized platelets
(31%-63%) occurred four days after the initial
appearance of organisms (figure 1).
Organisms appeared as basophilic inclusions
having one or more subunits (figure 2). Although

Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015

most platelets contained only one organism, two
or three organisms were not uncummon in a
single platelet. Individual subunits were not
easily discerned on Giemsa-stained smears be-
cause of the compact nature of the morula forms
of this organism (figure 2). Individual subunits
were more easily visualized, however, when dry
unfixed blood films were stained with new methy-
lene blue in 0.9% NaCI (figure 2). Organisms
were not observed in blood cell types other than
platelets.
During the first two to three days of the initial
parasitemic episodes, the platelet counts re-
mained within preinoculation and prepatent
ranges even though the percentage of parasitized
platelets increased from 0 to 2% to 14%. There-
after, platelet counts dropped rapidly, and mini-
mal platelet counts (2-15 X 103 / p.l) occurred
four to seven days after organisms were first ob-
served (figure I). Organisms were not present
on the days when minimal platelet counts were
recorded. Platelet counts increased to normal
values within three to four days after the mini-
mal counts were observed.
Organisms reappeared in the platelets of each
dog, although the maximal percentages of para-
sitemic platelets (5.5%-8%) were considerably
lower than those observed during the initial para-
sitized episodes. Dog no. 2 was sacrificed on day
20 before parasites had completely disappeared
so that pathologic examinations could be per-
formed at a time when organisms were present in
platelets in the circulation. The three remaining
dogs again became markedly thrombocytopenic
(3-9 X lOS platelets / p.l), and then platelet
counts rapidly increased. Dogs no. 3 and 4 (fig-
ure I) were each sampled through four addition-
al parasitemic episodes (maximal percentages
E
Figure 2. Top, a large platelet containing two com-
pact microorganisms on a Giemsa-stained blood film.
No subunits can be visualized. Bottom, two small
platelets, each containing a microorganism (arrows),
in a new methylene blue-stained wet preparation.
Erythrocytes (E) are unstained and appear as
"ghosts." Platelets (P) are lightly stained (both parts,
X2,400).
of parasitized platelets ranged from I % to 13%)
and associated thrombocytopenias (2-26 X lOS
platelets / JLl).
Parasitemias and thrombocytopenias appeared
to be "cyclic" in that they recurred within rela-
tively constant time periods. This periodicity
ranged from eight to 15 days (mean, 10.9 days)
when computed between maximal percentages of
parasitized platelets or from seven to 14 days
(mean, 10.5 days) when computed between mini-
mal platelet counts.
Changes in total leukocyte counts were not as
dramatic or as consistent as those seen in platelet
counts. Leukoctye counts usually decreased by
30%-50% in association with early parasitemias,
but no changes were apparent during the last
two parasitemias monitored in dogs no. 3 and 4.
Decreases in counts were not specific for anyone
leukocyte cell type, and total and absolute differ-
 Infectious Cyclic Thrombocytopenia 185

Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015

Figure 3. A, platelet contammg one microorganism with a single subunit (D); B, microorganism, containing
seven subunits (1-7), within a platelet; C, two microorganisms (arrows), each with a single subunit, in a platelet;
D, three microorganisms (1-3), each with three subunits, in a platelet (all parts, X20,OOO).
186 Harvey, Simpson, and Gaskin

ential leukocyte counts were never below normal
ranges [8].
The hematocrit decreased from 37% to 22% in
dog no. I during the course of the first two para-
sitemias and then increased back to preinocula-
tion values. It is unclear whether this anemia
was directly related to the presence of organisms
or thrombocytopenias, since only slight, transient
decreases (e.g., 44% down to 38%) were measured
in association with individual parasitemias in
the other three dogs. Erythrocyte indices were
always normal, and plasma protein concentra-
tions remained relatively constant.
surrounded by a single membrane, which more
or less conformed to the external surfaces of the
subunits that were surrounded by a double mem-
brane. The internal components of subunits con-
sisted of fibrillar material and small electron-dense
granules. Such granules appeared to be nucleo-
protein composed of both RNA and DNA since
they had an affinity for uranyl acetate [12].
Subunits were 350-1,250 nm in diameter and
oval-to-bean shaped; the bean shape was usually
associated with the divisionary process that trans-
formed a single subunit into two subunits. Binary
fission resulted from infolding of one side (fig-

Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015

Blood samples taken without anticoagulant ure 4) or opposite sides of the double limiting
during thrombocytopenias clotted rapidly. As membrane of subunits.
would be expected with a paucity of platelets, Necropsy findings. No gross pathologic le-
however, almost no clot retraction occurred dur- sions were noted at the time of necropsy. Of the
ing 24 hr at room temperature. several tissues examined by light microscopy,
Clinical signs. None of the dogs ever ap- changes were seen only in the lung. In this organ
peared clinically ill. A small amount of fresh there was a mild interstitial pneumonia charac-
blood was noted in the feces of dog no. 4 on one terized by fibrosis of the alveolar septal walls.
occasion when it was thrombocytopenic; other- There was also suggestive evidence of adherence
wise, no evidence of bleeding was observed even
though bleeding might be expected based on the
severity of the thrombocytopenias. (The bleed-
ing time of dog no. 3 was >30 min when the dog
was thrombocytopenic [2 X loa platelets/ILl]
compared with 2 min five days later when the
platelet count was 7.05 X 105 / ILL) The dogs
were essentially afebrile. Only two of the dogs
had moderately elevated rectal temperatures
(39.4 C-40.2 C) at the time of the initial para-
sitemias.
Splenectomized dogs (no. I and 4) had high
platelet counts before inoculation and between
parasitemias (figure I), and the recently splenec-
tomized dog (no. 4) had a moderately elevated
total white blood cell count. Otherwise, no differ-
ences in hematology, clinical signs, or pathology
were observed between splenectomized and in-
tact dogs.
Electron microscopy. Microorganisms were
found without difficulty in the cytoplasm of
platelets. Microorganisms contained one to eight
subunits (figure 3A and 3B), and up to three
microorganisms were present in a single platelet
(figure 3C and 3D). The percentage of the cyto-
plasm of the platelet occupied by microorgan-
isms was proportional to the number of micro-
Figure 4. Subunit division of a platelet-specific
organisms per cell and the number of subunits microorganism occurring from infolding (arrow) of
per microorganism. Each microorganism was the membrane surrounding the subunit (X20,OOO).
Infectious Cyclic Thrombocytopenia
of white blood cells to the endothelium of small
arteries.
Discussion
The microorganism reported herein appeared to
have a predilection for platelets, inasmuch as no
other blood cell types were found to be para-
sitized. It is suggested that this organism be clas-
sified in the order Rickettsiales since it does not
have a nucleus but appears to contain both RNA
and DNA based on staining with uranyl acetate
[12]. Subunits are very similar in size and struc-
ture to those seen in Ehrlichia canis infection of
dogs [13] and Anaplasma marginale infection of
cattle [12], the former organism being found in
leukocytes and macrophages and the latter in
erythrocytes. Subunits of this organism also bear
some resemblance to individual Haemobarto-
nella organisms [14-16] of various species (also
classified in the order Rickettsiales) found on
the surface of erythrocytes. Organisms in the
genus Haemobartonella (or Eperythrozoon),
however, have a single limiting membrane rather
than the double limiting membrane seen in sub-
units of E. canis, A. marginale, and the present
organism.
The morular forms of the platelet organisms
resemble those described in leukocytes of dogs
infected with E. canis except that they generally
consist of fewer subunits. (The smaller size of
platelets compared with that of leukocytes may
limit the size of morular forms within platelets.)
Isolates of E. canis vary in pathogenicity and the
predominant leukocyte type(s) containing or-
ganisms [17-19]. Organisms have not been re-
ported in platelets in canine ehrlichiosis.
Severely pathogenic strains of E. canis have
been demonstrated to be the causative agent of
a tick-borne disease called tropical canine pan-
cytopenia. After experimental inoculation with
E. canis, dogs develop evidence of disease within
10-15 days. During this initial febrile. period,
which persists for two to three weeks, they de-
velop anemia, leukopenia, and thrombocytopen-
ia. In some dogs, no further clinical illness oc-
curs, but others eventually develop marked pan-
cytopenia, hemorrhage, peripheral edema, ema-
ciation, and secondary bacterial infections 50-
100 days after infection [17].
Dogs in the present study were essentially
187
afebrile and did not become pancytopenic. How-
ever, they did become thrombocytopenic as has
been reported in canine ehrlichiosis. Thrombocy-
topenias occurred, however, as discrete cyclic
entities concomitant with parasitemic episodes,
rather than as prolonged thrombocytopenic
states as reported in ehrlichiosis. In addition, the
tissue infiltration with plasma cells that is charac-
teristic of E. canis infection [19] was not seen in
dogs with the platelet organisms. Indirect fluores-
cent antibody tests [20] of serum samples for E.
canis have thus far not conclusively suggested a
relatedness of the two organisms.
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015
Thrombocytopenias in the present study were
more severe than those reported in cattle in-
fected with an epicellular Eperythrozoon species
that appears to be platelet-specific [6]. As with
the Eperythrozoon organism of cattle, thrombo-
cytopenias in the present experiments in dogs
were associated with the appearance of parasites
in the circulation. In contrast to the single pro-
longed parasitemias and thrombocytopenias re-
ported in cattle, parasitemias and thrombocyte-
penias in the present study were discrete and
cyclic. There are other dissimilarities between
the two platelet parasites. The organism in dogs
occurs intracellularly and is usually composed of
multiple subunits in contrast to the epicellular
Eperythrozoon organism which does not have
subunits [6]. In addition, splenectomy is not re-
quired for infection with the agent in dogs.
Not only are the canine organisms incorpor-
ated into platelets, but clear evidence of intra-
cellular replication has been presented. The
mechanism of this incorporation is unknown. It
apparently occurs after platelet formation since
no organisms were observed in megakaryocytes
of bone marrow aspirates obtained on three oc-
casions when organisms were present in the
blood. The cyclic recurrence of microorganisms
in blood is not unique to the present agent but
has been noted with other blood parasites, e.g., in
feline haemobartonellosis [21]. The difference
between the magnitudes of the primary (first)
parasitemias (31%-63%) and of the subse-
quent parasitemias (1%-13%) is remarkable.
Thrombocytopenias following parasitemias with
only 1% infected platelets were as severe as those
following the initial parasitemias. It is suggested
that, whereas the initial thrombocytopenias may
develop primarily as a consequence of direct in-
188
jury to platelets by replicating parasites, immune-
mediated mechanisms of platelet removal be-
come more important in subsequent thrombo-
cytopenic episodes.
It is unclear why hemorrhage was not appar-
ent in the present study in dogs with severe
thrombocytopenias. Hemorrhage may not have
occurred because of the short duration of the
thrombocytopenias (usually only two or three
days). Human patients with immune thrombo-
cytopenic purpura bleed less often than people
with aplastic anemia having the same platelet
count [22]. It has been suggested that the constant
turnover of platelets in immune thrombocyto-
penic purpura affords greater vascular integrity
[22]. The rapid recovery of platelet counts after
parasitemias associated with increased thrombo-
poesis suggests a rapid turnover of platelets in
the present disorder.
The natural mode of transmission of this or-
ganism is unknown. It should be noted, however,
that the original dog was housed in a kennel for
several weeks prior to examination and that the
owner complained of a heavy tick infestation ac-
quired during this stay.
References
I. Clawson, C. C. Platelet interaction with bacteria. III.
Ultrastructure. Am. J. Pathol. 70:449--464, 1973.
2. Laird, H. M., Jarrett, 0., Anderson, I.. J., Jarret, W.
F. H. Electron microscopic detection of virus in fe-
line lymphosarcoma. J. Am. Vet. Med. Assoc. 158:
1109-1116,1971.
3. Fajardo, I.. F. Malaria parasites within platelets [ab-
stract]. Lab. Invest. 30:373, 1974.
4. Margolis, G., Kilham, I.. Rat virus infection of mega-
karyocytes: a factor in hemorrhagic encephalopathy?
Exp. Mol. Pathol. 16:326-340, 1972.
5. Hoover, E. A., Kociba, G. J. Bone lesions in cats with
anemia induced by feline leukemia virus. J. Natl.
Cancer Inst, 53:1277-1284, 1974.
6. Zwart, D., Leeflang, P., van Vorstenbosch, C. J. A. H. V.
Studies on eperythrozoon associated with bovine
thrombocytes. Zentralbl. Bakteriol. [Orig. A] 210:
82-102, 1969.
7. Garnham, P. C. C. Malaria parasites and other haemo-
Harvey, Simpson, and Gaskin
sporidia. Blackwell Scientific Publications, Oxford,
1966,p.1060-1065.
8. Schalm, O. W., Jain, N. C., Carroll, E. J. Veterinary
hematology. 3rd ed. Lea and Febiger, Philadelphia,
1975,p.26-38, 109.
9. Sabatini, D. D., Miller, F., Barrnett, R. J. Aldehyde
fixation for morphological and enzyme histochemical
studies with the electron microscope. J. Histochem.
Cytochem. 12:57-71, 1964.
10. Caulfield, J. B. Effects of varying the vehicle for Os04
in tissue fixation. J. Biophys. Biochem. Cytol. 3:827-
830,1957.
I I. Reynolds, E. S. The use of lead citrate at high pH as
an electron-opaque stain in electron microscopy. J.
Cell BioI. 17:208-212, 1963.
12. Simpson, C. F., Kling, J. M., Love, J. N. Morphologic
Downloaded from http://jid.oxfordjournals.org/ at East Carolina University on April 23, 2015
and histochemical nature of Anaplasma marginale.
Am. J. Vet. Res. 28:1055-1065,1967.
13. Simpson, C. F. Structure of Ehrlichia canis in blood
monocytes of a dog. Am. J. Vet. Res. 33:2451-2454,
1972.
14. Simpson, C. F., Love, J. N. Fine structure of Haemo-
bartonella bovis in blood and liver of splenectomized
calves. Am. J. Vet. Res. 31:225-231,1970.
15. Demaree, R. S., Nessmith, W. B. Ultrastructure of
Haemobartonella [ells from a naturally infected cat.
Am. J. Vet. Res. 33:1303-1308,1972.
16. Tanaka, H., Hall, W. T., Sheffield, J. B., Moore, D. H.
Fine structure of Haemobartonella muris as com-
pared with Eperythrozoon coccoides and Myco-
plasma pulmonis. J. Bacteriol, 90:1735-1749, 1965.
17. Buhles, W. C., Jr., Huxsoll, D. 1.., Ristic, M. Tropical
canine pancytopenia: clinical, hematologic, and sero-
logic response of dogs to Ehrlichia canis infection,
tetracycline therapy, and challenge inoculation. J.
Infect. Dis. 130:357-367, 1974.
18. Ewing, S. A., Roberson, W. R., Buckner, R. G., Hayat,
C. S. A new strain of Ehrlichia canis. J. Am. Vet. Med.
Assoc. 159:1771-1774, 1971.
19. Huxsoll, D. 1.., Hildebrandt, P. K., Nims, R. M., Walk-
er, J. S. Tropical canine pancytopenia. J. Am. Vet.
Med. Assoc. 157:1627-1632,1970.
20. Ristic, M., Huxsoll, D. 1.., Weisiger, R. M., Hilde-
brandt, P. K., Nyindo, M. B. A. Serological diagnosis
of tropical canine pancytopenia by indirect immuno-
fluorescence. Infec. Immun. 6:226-231, 1972.
21. Harvey, J. W., Gaskin, J. M. Experimental feline hae-
mobartonellosis. Journal of the American Animal
Hospital Association 13:28-38,1977.
22. Kitchens, C. S., Weiss, I.. Ultrastructural changes of
endothelium associated with thrombocytopenia.
Blood 46:567-578, 1975.