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Journal of Clinical Microbiology, April 2000, p. 1331-1338, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
New Ehrlichia Species Closely Related to
Ehrlichia chaffeensis Isolated from Ixodes ovatus
Ticks in Japan
Shin-ichiro
Shibata,1
Makoto
Kawahara,1
Yasuko
Rikihisa,*,2
Hiromi
Fujita,3
Yuriko
Watanabe,3
Chiharu
Suto,4 and
Tadahiko
Ito5
Nagoya City Public Health Research Institute,
Nagoya 467-8615,1 Ohara Research
Laboratory, Ohara General Hospital, Fukushima
960-0195,3 Department of Medical
Zoology, Nagoya University School of Medicine, Nagoya
466-8550,4 and Tokyo Metropolitan
Research Laboratory of Public Health, Tokyo
169-0073,5 Japan, and Department of
Veterinary Biosciences, College of Veterinary Medicine, The Ohio
State University, Columbus, Ohio 43210-10922
Received 6 October 1999/Returned for modification 8 December
1999/Accepted 13 January 2000
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ABSTRACT |
Seven Ehrlichia strains (six HF strains and one Anan
strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in
Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to
those of Anan strain, Ehrlichia chaffeensis (human
monocytic ehrlichiosis agent), and E. muris, respectively.
Partial GroEL amino acid sequencing also revealed that the six HF
strains had identical sequences, which were 99.0, 98.5, and 97.3%
identical to those of E. chaffeensis, the Anan strain, and
E. canis, respectively. All HF strains were lethal to mice
at higher dosages and intraperitoneal inoculation, whereas the Anan or
E. muris strain induced only mild clinical signs. Light and
electron microscopy of moribund mice inoculated with one of the HF
strains revealed severe liver necrosis and the presence of numerous
ehrlichial inclusions (morulae) in various organs. The study revealed
that members of E. canis genogroup are naturally present in
Ixodes ticks. HF strains that can cause severe illness in
immunocompetent laboratory mice would be valuable in studying the
pathogenesis and the roles of both cellular and humoral immune
responses in ehrlichiosis caused by E. canis genogroup.
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INTRODUCTION |
Ehrlichiae are obligatory
intracellular bacteria that infect monocytes/macrophages, granulocytes,
or platelets and cause a noncontagious, febrile systemic illness called
ehrlichiosis in humans and in some varieties of domestic and wild
animals (23-26). The severity of the disease varies from
asymptomatic seroconversion to death, and severe morbidity is
frequently documented. Ehrlichioses are now known as important emerging
vector-borne zoonoses in the United States (1, 2, 4, 5, 23-26,
33). Five different species of Ehrlichia (E. chaffeensis, human granulocytic ehrlichiosis [HGE] agent,
E. sennetsu, Venezuelan human Ehrlichia [VHE, a
strain of E. canis], and E. ewingii) are now
known to infect humans. Sennetsu fever in western Japan and in Malaysia
is caused by E. sennetsu (8, 17, 34). Human
monocytic ehrlichiosis (HME) in the United States is caused by E. chaffeensis (1), and asymptomatic human infection
with VHE occurs in South America (21). HME in Europe and
Africa (11, 18, 32) is probably caused by an Ehrlichia sp. closely related to E. chaffeensis.
HGE in the United States and Europe is caused by an ehrlichial organism
called HGE agent, which is closely related to E. equi and
E. phagocytophila (5, 10). HGE caused by
infection with another granulocytic Ehrlichia sp., E. ewingii (canine granulocytic Ehrlichia), was recognized
in Missouri last year (4).
Ehrlichia spp. are transmitted to humans by specific species
of infected ticks or trematodes from specific species of infected wild-animal reservoirs. For example, E. chaffeensis has been
most commonly identified in the Lone Star tick (Amblyomma
americanum) (3), and white-tailed deer are considered
to be the major reservoir of E. chaffeensis (6,
15). The HGE agent has been found in the deer tick (Ixodes
scapularis) (20, 31), and white-footed mice are
considered to be the major reservoir of the HGE agent in northeastern
and midwestern United States (31). Most Ehrlichia spp. characterized so far were isolated from domestic animals or
humans. Only a few Ehrlichia spp. have been isolated from
wild animals or vectors. Examination of Ehrlichia spp. in
vectors and wild animals would provide an understanding of natural
distribution and maintenance of ehrlichial organisms, as well as the
diversity and evolution of ehrlichial populations. Such a study would
provide a risk assessment for acquiring ehrlichial infection in
particular geographic regions and, therefore, would facilitate
proactive preventive measures. In 1983, Kawahara et al. isolated an
infectious agent inducing splenomegaly in laboratory mice from a wild
mouse (Eothenomys kageus) caught in central Japan. This
organism was shown to be closely related to Ehrlichia canis
by morphological and antigenic analysis (12), and 16S rRNA
gene sequencing revealed it to be most closely related to E. chaffeensis. Since it is sufficiently distinct from any known
Ehrlichia spp., it was designated E. muris (35). E. muris has been isolated from two
additional species of wild mice in Metropolitan Tokyo and from
Haemaphysalis flava ticks in Japan (14) (Fig.
1). Seroepidemiologic data suggested the
exposure of humans and various wild animals to E. muris or related species in Japan (14).
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FIG. 1.
Geographic region where Ehrlichia spp. were
isolated from ticks or wild mice. Tick isolates are boxed. An asterisk
indicates an isolate used for this study. Letters designate sources as
follows: a, Fujita and Watanabe (7);
b, Kawahara et al. (12); and c,
Kawahara et al. (14).
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Fujita and Watanabe (7) isolated 14 Ehrlichia-like agents from Ixodes ovatus ticks
collected in two northern prefectures in Japan from 1983 to 1994 (Fig.
1). In the present study, we characterized five isolates collected from
1993 to 1994, which were reported by Fujita and Watanabe
(7), and two additional tick isolates that have not been
reported previously. To accomplish this analysis, we examined
genetic, antigenic, and ultrastructural features and concluded that the
strains are most closely related to E. chaffeensis, followed
by E. muris and E. canis. Histopathologic observations in infected immunocompetent mice suggests that these new
strains would be valuable in studying disease mechanisms and the role
of immune responses in ehrlichiosis caused by the E. canis genogroup.
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MATERIALS AND METHODS |
Ehrlichiae isolated from I. ovatus
ticks.
Five isolates (HF565, HF568-1, HF568-2, HF639-2, and HF642)
were reported previously (7), and two isolates (HF652 and
Anan) were obtained in the present study. HF652 was isolated from
I. ovatus collected in Aomori Prefecture in 1994, and Anan
was isolated from I. ovatus collected in Tokushima
Prefecture in 1994 (Fig. 1). All ticks were collected from vegetation
with a standard 1-m2 flannel flag. Isolation of
Ehrlichia spp. was done as described elsewhere
(7). Briefly, ticks were soaked for 10 min in 70% ethanol
with 0.1% povidone-iodine and then rinsed with phosphate-buffered saline (PBS, pH 7.2) with 0.5 to 1.0% calf serum (GIBCO, Grand Island,
N. Y.). Of the pooled ticks, two to six
either male or femalewere ground up with a depression slide glass and a glass pestle
(Iuchi Co., Ltd., Osaka, Japan) or with a mortar and pestle in
sucrose-phosphate-glutamate (0.0038 M
KH2PO4-0.0072 M
K2HPO4-0.0049 M L-glutamate-0.218
M sucrose [pH 7.2]) at 0.3 ml per tick. For isolation of infectious
agents, 0.3 ml of the homogenate of each pool was inoculated
intraperitoneally into one 6-week-old female ddY mouse (Funabashi Farm,
Shizuoka, Japan). These isolates had been passaged through mice once or twice.
Analysis of 16S rRNA and GroEL sequences of strains.
DNA was
extracted from the spleens harvested from mice infected with strains
HF565, HF568-1, HF568-2, HF639-2, HF642, HF652, or Anan by using a
blood kit (Qiagen, Inc., Valencia, Calif.). PCR amplifications and
nucleotide sequencing of total 16S rRNA genes were performed as
previously described (14).
PCR amplification and nucleotide sequencing of
groEL genes
in DNA from the spleens of mice infected with HF and Anan isolates
were
performed by the method of Sumner et al. (
30). The primer
pairs used were HS43 and HS45 (
30), Cha792u
(5'-GGTGATGGAACAACTACATG-3')
and Cha1429d
(5'-CCWARCATRTCTTTTCTTCT-3'), and Cha1293u
(5'-GARGTDDARGGTGAAGC-3')
and Cha1770d
(5'-TTCAACAGCAGCTCTAGTTG-3'), which were designed
based on
E. chaffeensis and HS43-HS45-amplified products. The
PCR
products were sequenced by using the PCR
primers.
Phylogenetic analysis of DNA and amino acid sequences.
Sequence data were prepared for analysis by using the AutoAssembler
version 1.4 (Perkin-Elmer, Norwalk, Conn.). The corrected levels of
divergences of nucleotide of the 16S rRNA gene and deduced amino acid
sequences of GroEL were calculated by using the Genetic Information
Processing Software GENETYX-WIN version 3.0 (Software Development Co.,
Tokyo, Japan) on a Power Mac 8500. A dendrogram was constructed by
using the UPGMA method and data from the distance matrix.
Clinical signs, relative spleen size, and indirect fluorescence
antibody (IFA) titer against E. muris.
The 10%
homogenates of spleens prepared from each mouse infected with one of
the six HF strains at days 9 to 10 postinfection (p.i.), just before
death, at a 1:10 dilution and the 10% homogenate prepared from the
mouse infected with the Anan strain at day 10 or 15 p.i. without
dilution were inoculated intraperitoneally or subcutaneously at 0.2 ml/mouse into five BALB/c male mice (8 weeks old). As positive controls
the homogenates from mice infected with three strains of E. muris AS145 (type strain [12]), I-268 (14), and NA-1 (14) at 10 days p.i. were
inoculated into five mice each. The spleen homogenates used as inocula
contained similar levels of ehrlichial DNA based on the band intensity
of the PCR products, except for that of the Anan isolate. The band
intensity of the Anan isolate in the 10% spleen homogenate
corresponded to an ~103 dilution of isolates from HF
strains. To assess the ehrlichial amount in the inoculum, the PCR
amplification of each of the strains was performed by using a thermal
cycler (Sankojyunyaku Co., Ltd., Tokyo, Japan) and GeneAmp reagents
(Takara, Kyoto, Japan). A pair of primers, EC9
(5'-AAGGATCCTACCTTGTTACGACTT-3') and EC10
(5'-AATCTAGATTAGATACCCTDGTAGTCC-3', in which D = A, T, or G) were used based on the bacterial sequence of 16S rRNA
(1). Each sample was amplified for 3 cycles at 94°C (1 min), 48°C (2 min), and 66°C (1 min 30 s), followed by 94°C
for 9 min and 25 cycles at 88°C (1 min), 52°C (2 min), and 68°C
(1 min 30 s). Products were electrophoresed through a 1.5% agarose gel to assess amplification efficiency. A 733-bp fragment was
obtained after the amplification of DNA templates from all isolates.
The clinical signs of inoculated mice were observed daily until death
or
20 days p.i. The relative spleen indexes (in grams
per 100 g
of body weight) were compared among all mice inoculated
intraperitoneally on days 8 and 10 p.i. Antibody titers were
compared
for all mice inoculated subcutaneously on day 20 p.i.
Antibody
titers against
E. muris, the HGE agent, and
E. sennetsu were measured
by the IFA test (
12,
29). Various tissues, including the liver,
spleen, thymus, and
bone marrow, were collected at days 7 to 10
from mice inoculated
intraperitoneally with HF565 or Anan strain,
fixed in 10% buffered
formalin solution (pH 7.4), and embedded
in paraffin. Sections were
stained with hematoxylin-eosin and
Giemsa.
Electron microscopy.
The spleen and liver samples from mice
intraperitoneally inoculated with strain HF565 at days 7 and 9 p.i. or with Anan strain at day 10 p.i. were cut into 1-mm-thick
cubes and fixed and processed as described elsewhere (12).
Ultrathin sections were stained with uranyl acetate and lead citrate,
and the stained sections were examined with a Philips model 300 electron microscope at 60 kV.
Nucleotide sequence accession numbers.
The GenBank
nucleotide sequence accession numbers for the 16S rRNAs used for
comparison in this study are as follows: E. chaffeensis, M73222; E. canis, M73221; E. ewingii, M73227;
E. sennetsu, M73225; E. muris, type strain AS145,
U15527; strain I-268, AB013008; and strain NA-1, AB013009. The
nucleotide sequences of strain HF565 and strain Anan have been
deposited in the GenBank data library under accession numbers
AB024928 and AB028319, respectively. The accession numbers of
groEL sequences used for sequence comparisons are Z15160 for
Bartonella bacilliformis, U13638 for Cowdria
ruminantium, L10917 for E. chaffeensis, U88092 for
E. sennetsu, X07850 for Escherichia coli, U96728 for HGE agent, U96727 for E. equi, U96731 for E. canis, and U96729 for E. phagocytophila. Nucleotide
sequences of strain HF-565 and strain Anan have been deposited in the
GenBank data library under accession numbers AB032712 and AB032711, respectively.
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RESULTS |
16S rRNA gene and partial GroEL amino acid sequences and
phylogenetic analysis.
All ehrlichial strains isolated from wild
mice or ticks in Japan, including three strains of E. muris
AS145 (type strain, 11), I-268 isolated from an Apodemus
speciosus wild mouse in Metropolitan Tokyo (14), or
NA-1 isolated from a H. flava tick in Aichi Prefecture (14), are shown in Fig. 1. A total of 16 strains were
isolated from adult I. ovatus: 12 strains in Fukushima
Prefecture from 1983 to 1994, 3 strains in Aomori Prefecture from 1984 and 1994, and 1 strain in Tokushima Prefecture in 1994 (Fig. 1)
(7). Four Fukushima strains (HF565, HF568-1, HF568-2, and
HF639-2), two Aomori strains (HF642 and HF652), and one Tokushima
strain (Anan) were examined in the present study.
Of the 1,449-bp 16S rRNA gene sequences compared, all HF strains had
identical base sequences and between Anan and HF strains,
only three
bases were different (Table
1). Anan and
HF strains
belonged to the
E. canis genogroup and were far
apart from the
E. phagocytophila (the HGE agent) and
E. sennetsu genogroups (Table
1 and Fig.
2). The HF strains were closest to
E. chaffeensis,
even more so than
E. muris: HF
strains and
E. muris differ from
E. chaffeensis
by 19 and 29 bases, respectively (Table
1).
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FIG. 2.
Phylogenetic relationships between Anan, HF, and other
Ehrlichia strains based on 16S rRNA gene sequence
comparisons. HF strains included HF565, HF568-1, HF568-2, HF639-2,
HF642, and HF652.
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Of the 409 deduced amino acid sequences of GroEL compared, all six HF
strains had the identical amino acid sequence (Table
2). All strains belonged to
E. canis genogroup and were far from
the
E. phagocytophila
(the HGE agent) or
E. sennetsu genogroup
(Table
2 and Fig.
3). HF strains were closest to
E. chaffeensis,
followed by the Anan strain and
E. canis
in the dendrogram (Fig.
3). Between HF strains and
E. chaffeensis, four amino acids were
different, and between HF and
Anan strains six amino acids were
different (Table
2).
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FIG. 3.
Phylogenetic relationships generated with UPGMA based on
an alignment of the first 409 amino acid sequences of GroEL of HF and
Anan strains and other members of the tribe Ehrlichieae.
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Antibody reactivity.
Antibody titers against E. muris were examined by the IFA test in the sera collected on day
20 p.i. from mice inoculated subcutaneously. The titers of sera
from mice inoculated with all I. ovatus isolates were
comparable with three strains of E. muris: AS145, I-268, and
NA-1 (Table 3). There was no
cross-reactivity of these sera with E. sennetsu or the HGE
agent. Because E. muris, E. chaffeensis, E. canis, VHE, and E. ewingii are antigenically highly
cross-reactive to each other (1, 2, 4, 12, 14, 21, 23-26),
this result is in agreement with the 16S rRNA gene and GroEL sequencing data showing that all I. ovatus isolates examined belong to
the E. canis genogroup rather than the E. phagocytophila or E. sennetsu genogroup.
Mouse pathogenicity.
When ca. 10% spleen of the homogenate of
mice containing each strain was intraperitoneally inoculated, starting
ca. day 7 p.i., all mice inoculated with six HF strains developed
clinical signs of ruffled fur, inactivity, anorexia, dehydration, and
weight loss, and they died from days 8 through 10 p.i. (Table 3).
The lethality of the HF strains was dependent on the dosage and route of inoculation. With a dilution of up to 102 of the
inoculum, all mice inoculated intraperitoneally died at days 8 to
10 p.i., but at a 104 dilution all mice survived. With
the subcutaneous route, two of five mice died on days 8 to 10 p.i., but with >10-fold dilutions of inoculum all mice survived for
more than 20 days p.i., at which time the experiment was terminated.
The mice inoculated intraperitoneally with the Anan strain without
dilution developed mild clinical signs similar to those
inoculated with the E. muris strains (12, 13).
Mice inoculated subcutaneously with the Anan isolate did not develop
any significant clinical signs. None of mice inoculated intraperitoneally with the three strains of E. muris AS145,
I-268, or NA-1 died during the 20-day p.i. period (Table 3). By PCR, ehrlichial DNAs present in 10% of the spleen homogenates were similar
among all strains, including E. muris strains, except the
Anan strain. The Anan strain in the spleen was approximately 1/103 of the HF565 strain in the spleen. Lack of death with
the Anan strain was, however, not caused by small amounts of organisms in the inoculum. When the Anan strain at the same DNA level as that of
the HF565 strain was intraperitoneally inoculated into mice, it did not
kill them (data not shown).
At necropsy, mice inoculated with all strains developed splenomegaly.
When spleen sizes were compared among intraperitoneally
inoculated mice
on days 8 to 10 p.i., the degrees of splenomegaly
were greater in
the following order: I-268, NA-1,
E. muris (AS145)
> Anan > all HF strains (Table
3). The mean relative spleen
size
of uninfected mice was 0.42 ± 0.04 (
n = 5);
therefore, there were
seven- to twofold increases in the spleen size.
The spleens were
dull dark red and tender in HF strain-inoculated mice.
In contrast,
the tissues were less dark and firm in mice inoculated
with Anan
or
E. muris strains.
Light and electron microscopy of organs of infected mice.
By
light microscopy of paraffin sections numerous dark-blue (Giemsa)- or
purple (hematoxylin-eosin)-stained cocci in round inclusions (morulae)
or by light microscopy of Epon-embedded 1-µm sections light-blue
(toluidine blue)-stained organisms could be visualized. These organisms
were frequently observed in the cytoplasms of mononuclear cells in the
lumen and along the wall of small blood vessels in the spleen, liver,
thymus, lung, bone marrow, and large intestine of mice
intraperitoneally inoculated with strain HF565 on days 7 and 9 p.i. (Fig. 4). Prominent neutrophilic inflammatory cell infiltration was not evident in any of these organs.
In the spleen, the red pulp was expanded, the white pulp was
disorganized, and the follicles were not significantly activated. Thymic severe lymphoid depletion as seen in E. risticii-infected mice (27) was not evident in the mice
infected with HF strain. The liver showed severe necrosis, especially
around the central veins. Morulae were not seen in the necrotic area.
In contrast, morulae were not detectable in the spleen of Anan
strain-infected mice on days 10 and 15 p.i. The spleens of mice
inoculated with Anan strain had increased cellularlity and
well-developed follicles even at day 7 p.i. (data not shown).
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FIG. 4.
Light micrographs of paraffin-embedded section of the
thymus (A) and the lung (B) from the mice intraperitoneally inoculated
with HF565 strain at day 9 p.i. Note several morulae packed with
many ehrlichial organisms (colonies of ehrlichiae) along the capillary
(arrows) (A) [magnification, ×1,070]) and in the capillary of the
alveolar wall (arrows) (B) [magnification, ×1,700]). Hemtoxylin and
eosin staining was used.
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Under an electron microscope, numerous ehrlichial organisms were
detected in membrane-bound inclusions in the cytoplasm of
monocytes,
macrophages, and eosinophils but not in neutrophils
in the red pulp of
the spleen or in Kupffer cells in the liver
from mice inoculated with
strain HF565 (Fig.
5).
The organisms
were generally round, but some were pleomorphic and
variable in
size, ranging from 0.4 to 1.0 µm in length and 0.2 to 0.7 µm in
diameter. Each organism was bound by two membranes, and the
outer
membranes were ruffled (Fig.
5). Significant amounts of ribosomes
and a fine meshwork of DNA strands were evident (Fig.
5). As noted
previously in
E. canis inclusions in DH82 cells
(
23) and in
various
E. chaffeensis strains
(
25), the intramorular matrix
contained tiny fibrillar
materials in some morulae (Fig.
5). Intramorular
tubules of
approximately 25 nm in diameter, which are found in
the
inclusions of several strains of
E. chaffeensis in DH82
cells
(
25), were occasionally seen (one is indicated Fig.
5B). As
noted in
E. chaffeensis (
25), occasional
association of mitochondria
with ehrlichial inclusions was seen (Fig.
5A and B). These findings
are in agreement with the 16S rRNA and
groEL gene sequencing data
showing that HF565 strain belongs
to
E. canis genogroup. Ehrlichial
organisms were not
detectable in the spleens of Anan strain-infected
mice by electron
microscopy.
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FIG. 5.
Electron micrographs of morulae of HF565 in the
cytoplasm of a monocyte (A) and an eosinophil (B) in the blood vessel
in the spleen and in a Kupffer cell in the liver (C) from the mouse at
day 7 p.i. Note the numerous pleomorphic coccobacilli enveloped in
two layers of membranes embedded in a fine filamentous matrix in the
membrane-bound inclusion in panel A. An intramorular tubule is
indicated by the arrow in panel B. The inclusions are tightly packed
with ehrlichiae without intramorular space in the Kupffer cells adhered
to the endothelial layer lining the sinusoid. Magnifications: ×24,200
(A), ×21,000 (B), and ×16,400 (C).
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DISCUSSION |
16S rRNA genes and GroEL amino acid sequences, antigenic
cross-reactivities, and ultrastructural features indicate that our I. ovatus isolates in Japan belong to the E. canis genogroup and are most closely related to E. chaffeensis. E. chaffeensis has been found so far
primarily in Amblyomma americanum and, less frequently, in
Dermacentor variabilis in the United States (3). A small number of the I. scapularis ticks tested were
negative for E. chaffeensis (3). Most
Ehrlichia spp. found in Ixodes spp. in the United
States and Europe are Ehrlichia spp. belonging to E. phagocytophila, E. equi, and the HGE agent genogroup
(24). However, a partial 16S rRNA gene sequence identical to
that of E. muris was recently found in Ixodes
persulcatus ticks in Perm, Russia (22). Therefore,
Ixodes spp. can be naturally infected by two distinct
ehrlichial genogroups. I. ovatus is a common tick found
throughout Japan (38). The adult-stage tick bites humans and
large animals, and the nymphal- and larval-stages tick bite small
mammals such as rodents (38). Whether, like E. muris, wild mice are reservoirs of these I. ovatus
isolates is yet unknown. Two species of ticks have so far been
found infected with Ehrlichia spp. in Japan. We previously
isolated E. muris from H. flava ticks (14). According to Fujita and Watanabe (7), the
ehrlichial infection rate for I. ovatus (16 of 439 ticks
examined) is higher than for H. flava (0 of 351 ticks examined).
By electron microscopy HF565 organisms, which belong to the
monocytotropic ehrlichiae, were detected in eosinophils. It is unlikely
that I. ovatus was coinfected with granulocytotropic Ehrlichia sp., since mouse neutrophils were not infected and
our serologic and gene sequencing data did not reveal any contamination of granulocytotropic Ehrlichia sp. in the inoculum.
Ehrlichial organisms were previously seen in neutrophils in the blood
of HME patients (16, 19). Therefore, although this is a rare event and monocytes are the primary hosts, in severe infections some
granulocytes are infected with the E. canis genogroup.
Whether this occurs with granulocytes from uninfected healthy hosts in vitro or due to alterations that occur in granulocytes in infected hosts is unknown.
Ehrlichia spp. can be subdivided into three genetically
distinct groups (genogroups). Two ehrlichial genogroups have been isolated in Japan to date. E. sennetsu is the first
ehrlichial organism discovered in Japan. It was isolated from the blood
of patients with "Hyuga fever" or "Kagami fever," which was
endemic in Kyushu, Southern Japan, in the 1950s (8, 17). SF
agent, which belongs to E. sennetsu genogroup
(36), had originally been isolated in Japan in 1962 from
Stellantchasmus falcatus metacercarial parasites on gray
mullet fish in Kyushu (9). The second genogroup organisms isolated were the E. muris strains (12,
14) and I. ovatus strains described here. The third
E. phagocytophila genogroup has not yet been identified in Japan.
HF strains, when inoculated intraperitoneally, were more virulent in
immunocompetent laboratory mice than any other ehrlichial strains we
examined. Because all six strains obtained at different times in
different geographic regions had similar levels of virulence, it is
unlikely that the viruses or other agents are contaminated in all of
these isolates. We have tried to cultivate HF and Anan strains by using
various cell lines. We have not cultivated any of them yet, but we have
not found any contamination by bacteria, viruses, or parasites in these
isolates. Haemobartonella or Eperythrozoon spp.
were not detectable on a blood smear or by a PCR based on the 16S rRNA
gene sequences we determined (28). Therefore, it is unlikely
that other contaminating agents are responsible for the virulence of
the HF strains.
Of significance is the striking difference in mouse pathogenicity
between HF strains and the Anan strain, despite the closeness of their
16S rRNA gene sequences. Massive ehrlichial proliferation was evident
in immunocompetent mice with HF stains, whereas proliferation of the
Anan strain-like E. muris appears to be held in check. As
previously noted with E. muris infection of mice
(13), this may be due to active immune stimulation with the
Anan strain, as evidenced by increased cellularlity and follicle
stimulation in the spleen. On the other hand, with lethal dosages of HF
strain, the spleen structure was disorganized and follicle stimulation was not evident. With the HF strain widespread prominent liver necrosis
similar to that previously noted in fatal HME (D. H. Walker,
J. P. Taylor, J. S. Blie, and C. Dearden, Abstr. 89th Annu.
Meet. Am. Soc. Microbiol. 1989, abstr. D76, p. 95, 1989) or in
SCID/beige mice experimentally infected with E. chaffeensis (37) was seen. In connection with this observation, one of
the characteristic laboratory findings of HME as well as with HGE is
increased liver enzyme activities. What gene products of HF strain are
responsible for this fulminant infection in immunocompetent mice is
unknown. These two isolates, i.e., HF and Anan, the closest relatives
of E. chaffeensis, may provide a valuable comparative immunocompetent mouse disease model for understanding pathogenesis and
roles of immune responses in ehrlichiosis caused by the E. canis genogroup.
| |
ACKNOWLEDGMENTS |
We thank Fumihiko Mahara for kindly providing accommodations for
tick collection in Anan City, Tokushima, Japan.
A part of the study was supported by the grant RO1 AI30010 from the
National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1092. Phone: (614) 292-5661. Fax: (614) 292-6473. E-mail: rikihisa.1{at}OSU.edu.
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Abstract
Introduction
