帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
Serol ogi cal evi dence of i nfecti on of Anapl asm a
and Ehrl i chi a i n dom esti c ani m al s i n Xi nj i ang
Uygur Autonom ous Regi on area, Chi na
Chahan, Bayi n, J i an, Zi j i an, Xuan, Xuenan, Sato,
Yuki ta, Kabeya, H i denori , Tuchi ya, Kotaro,
Itam oto, Kazuhi to, O kuda, M asaru, M i kam i ,
Takeshi , M aruyam a, Soi chi , Inokum a, H i sashi
CitationVETERIN ARY PARASITO LO GY, 134(3-4): 273-278
URLhttp: //i r. obi hi ro. ac. j p/dspace/handl e/10322/742
Veterinary Parasitology Revised MS
25 July 2005
Serological Evidence of Infection of Anaplasma and Ehrlichia
in Domestic Animals in Xinjiang Uygur Autonomous Region Area, China.
Bayin Chahan1), Zijian Jian1), Xuenan Xuan２), Yukita Sato３), Hidenori Kabeya３), Kotaro
Tuchiya４), Kazuhito Itamoto５), Masaru Okuda５), Takeshi Mikami3), Soichi Maruyama３),
1) Veterinary College, Xingjiang Agricultural University, Urumqui, China, 2) National
Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary
Medicine, Inada, Obihiro 080-8555 Japan, 3) College of Bioresource Science, Nihon
University, 1866 Kameino, Fujisawa 252-8510 Japan, 4) Nippon institute for Biological
Science, Ome, Tokyo 198-0024, Japan, 5) Faculty of Agriculture, Yamaguchi University,
1677-1 Yoshida, Yamaguchi 753-8515 Japan, 6) Faculty of Veterinary Medicine, Obihiro
University of Agriculture and Veterinary Medicine, Inada, Obihiro 080-8555 Japan
*: Corresponding to Hisashi INOKUMA,
Faculty of Veterinary Medicine, Obihiro University of Agriculture & Veterinary Medicine,
Inada, Obihiro 080-8555, Japan
TEL/FAX [+81]-155-495370, e-mail: email@example.com
Serological methods were utilized to detect Anaplasma and Ehrlichia infection in
domestic animals in Xinjiang Uygur Autonomous Region, China. By using an indirect
immunofluorescence assay, antibodies that reacted with A. phagocytophilum and E.
chaffeensis were detected mainly in ruminants kept on pastureland in Altai, Ili and Kashgar
area. Antibody titers up to 1:320 were recorded. These results indicate that ruminants kept
in these areas may be infected with some species of Anaplasma and Ehrlichia.
Keywords. Anaplasma, Ehrlichia, domestic animals, seroepidemiology, Xinjiang Uygur
Autonomous Region, China
Ehrlichioses are important vector-borne diseases in both humans and animals. Both
Anaplasma and Ehrlichia spp. are known to be transmitted by arthropod ticks and are
distributed worldwide (Dumler et al., 1998). The genus Anaplasma includes A. marginale, A.
centrale, A. ovis, A. platys, A. phagocytophilum and some unidentified species closely related
to those pathogens. The genus Ehrlichia includes E. canis, E. ewingii, E. chaffeensis, E.
muris, E. ruminantium, and some additional new Ehrlichia species. A. phagocytophilum and
E. chaffeensis are two major zoonosis pathogens mainly reported in the United States and
European countries (Foley et al., 2004; Parola, 2004). A. phagocytophilum can cause
prevalent diseases in humans, ruminants and horses, and E. chaffeensis in both humans and
dogs. Recently, both agents have also been reported in eastern Asia, including China and
Korea (Cao et al., 2000; Cao et al., 2003; Kim et al., 2003). In China, DNA fragments of A.
phagocytophilum have been detected in Ixodes persulcatus ticks in Heilongjiang Province in
northeastern China (Cao et al., 2003). E. chaffeensis DNA were also detected by PCR from
Haemaphysalis yeni and Amblyomma testudinarium in southern China (Cao et al., 2000).
However, there has been little information available on ehrlichiosis in the western part of
China. Xinjiang Uygur Autonomous Region is located in the western most area in China.
The region has a cold and dry climate with high mountains and wide deserts. The animal
grazing of ruminants on pastureland is one of the main industries of Xinjiang. Horses and
donkeys are also important animals for use in transportation in this area. Rickettsia sibilica
is the only known rickettsial pathogen that causes spotted fever in humans in Xinjiang (Ai et
al., 1979; Fan et al., 1987), but it is not clear whether other tick-borne rickettsial diseases exist.
The aim of this study was to determine whether pathogens of Anaplasma and Ehrlichia
distribute in Xinjiang Uygur Autonomous Region. Thus the sero-prevalence of antibodies
against Anaplasma and Ehrlichia in domestic animals, including cattle, sheep, goats, horses
and donkeys in this area were screened by using indirect immunofluorescence (IFA) test for A.
phagocytophilum and E. chaffeensis.
2. Materials and Methods
2.1 Sera from animals
Sera were collected from 146 cattle, 134 sheep, 133 goats, 85 horses and 100
donkeys in Xinjiang Uygur Autonomous Region from April to August in 2004. Histories
and clinical symptoms of each animal were not recorded. Three areas, Altai, Ili and Kashgar,
were selected for the survey (Fig.1). Altai is situated in the northern part of Xinjiang, and is
bounded by Russia and the People's Republic of Mongolia. It is just south-west of the Altai
Mountains. Ili is situated at the north-west border of Xinjiang, and is bounded by the
Kazakhstan Republic, Russia. It is also north of the Tianshan Mountains. Kashgar is at the
west end of Xinjiang, bordering the Taklamakan desert in the east and the Kunlun Range in
the south. It is also the eastern neighbor of Kyrgys and Tajikistan. The numbers of sera
from each area and type of animal are shown in Table 1. Samples were stored at –20°C until
2.2. IFA test
Antigens for IFA test were kindly given by Dr. P. Brouqui (Unité des Rickettsies,
Université de la Méditerranée, Marseille, France). A. phagocytophilum (HGE agent Webster
strain, originally supplied by Dr. J. S. Dumler, The Johns Hopkins University School of
Medicine, Baltimore, MD, USA) and E. chaffeensis (Arkansas strain, originally supplied by
Dr. J. E. Dawson, Center for Diseases Control and Prevention, Atlanta, GA, USA) were used
as antigens in the IFA test as previously described (Brouqui et al., 1994). Sera from mice
that were experimentally infected with A. phagocytophilum and E. chaffeensis were used as
positive controls. Sera from healthy animals kept in Japan were used as negative controls.
Sera were screened at a 1:20 dilution in phosphate-buffered saline (pH 7.4), Tween 0.5%
(PBST) and an optimized dilution (1:160 to 1:200) of fluorescein isothiocyanate-labelled
anti-IgG conjugate (anti-cattle IgG; ICN Pharmaceuticals Inc., U.S.A., anti-sheep IgG; ICN
Pharmaceuticals Inc., U.S.A., Capple, anti-goat IgG; ICN Pharmaceuticals Inc., U.S.A.,
anti-horse IgG, MP Biomedicals, Inc., U.S.A., or anti-donkey IgG; Santa Cruz Biotechnology,
U.S.A.) in PBST was used as the second antibody. Reactive antibodies were then detected
using a fluorescence light microscope. Antibody levels of test samples were determined by
comparison with the appropriate positive and negative controls. Those samples that reacted
with any of the antigens at the screening dilution were then titrated using serial twofold
dilutions to determine end titers.
3. Results and Discussion
The results are summarized in Table 2. A total of 7 cattle serum samples among 47
(14.9 %) in Altai, 6 among 50 (12.0 %) in Ili, and 2 among 49 (4.1 %) in Kashgar reacted
with at least one of the antigens at a dilution of 1:40 or more. Dual positivity was
occasionally seen, but most samples reacted more strongly with one of the two antigens. In
Altai, all of the 7 positive cattle reacted with E. chaffeensis with titers of 1:40 to 1:320, and
showed weak reaction with A. phagocytophilum with titers of 1:20 or less. Five cattle serum
samples in Ili showed higher titers against E. chaffeensis (1:40 to 1:160), while 1 showed a
higher titer against A. phagocytophilum (1:80). In contrast, the 2 positive cattle in Kashgar
showed higher titers against A. phagocytophilum (1:40 and 1:160) than those against E.
chaffeensis.A total of 6 sheep serum samples among 37 (16.2 %) in Altai, 11 among 50
(22.0 %) in Ili and 8 among 47 (14.9 %) in Kashgar, reacted with A. phagocytophilum or E.
chaffeensis at a dilution of 1:40 or more. In Altai, 3 of the 6 sheep sera showed higher titers
against A. phagocytophilum (with titers of 1:40 to 1:160) than those against E. chaffeensis,
while the other 3 samples showed the same titers (of 1:40 or 1:80) against A.
phagocytophilum and E. chaffeensis. In Ili, 4 sheep samples showed higher titers against E.
chaffeensis (1:80 to 1:160), and 1 against A. phagocytophilum (1:40), while the other 6
showed the same titers against both antigens. In Kashgar, 3 positive sheep sera showed
higher titers against A. phagocytophilum (1:40, 1:80 and 1:320), 3 showed higher titers
against E. chaffeensis, and the other 2 showed equal titers (of 1:40 and 1:80) against both
antigens. None of the goat sera in Altai showed any positive reaction, while a total of 3 goat
serum samples among 50 (6.0 %) in Kashgar and 1 among 33 (3.0 %) in Ili reacted with A.
phagocytophilum or E. chaffeensis at a dilution of 1:40 or more. In Ili, the only positive
sample showed a higher titer against E. chaffeensis, with titer of 1:40. In Kashgar, 2 positive
goat sera showed higher titers against A. phagocytophilum (1:40 and 1:80) and 1 showed a
higher titer against E. chaffeensis (1:40).
In the present study, antibodies that reacted with A. phagocytophilum and E.
chaffeensis were detected in ruminants in Xinjiang. However, the relationship between
pathogenesis and antibodies against these agents was not analyzed, because the histories and
clinical symptoms were not recorded in this study. In Altai, cattle showed higher titers
against A. phagocytophilum, while sheep showed higher titers against E. chaffeensis. This
may reflect the differences of location of where the examined animals were kept. It was
impossible to examine the existence of A. phagocytophilum and E. chaffeensis in these areas,
because cross reaction of antibodies is commonly seen for antigens among the same genus.
The positive reaction might have resulted from infection of species closely related to A.
phagocytophilum and E. chaffeensis. Higher titers may be associated with multiple exposure
to individual animals or recent exposure, although some younger animals also showed higher
titers. In Xinjiang, most ruminants are kept on pastureland, and are usually infested with
many ticks from spring to autumn. Ticks may transmit the ehrlichial pathogens to animals.
All the horse serum samples were obtained in Altai, and none of there sera reacted
with any of the antigens. The only positive serum of donkey was obtained from an animal in
Kashgar. The titer against A. phagocytophilum was 1:40. Most of the horses and donkeys
examined in this study were not kept on pastureland, but lived near the farm houses and were
used for transportation. Thus, tick infestation of horses and donkeys is less likely than that
Recently, several new ehrlichial species were detected by molecular methods or
isolated around China. Ehrlichia muris and a new Ehrlichia species closely related to E.
chaffeensis were isolated in Japan (Shibata et al., 2000; Wen et al., 1995). E. muris DNA has
also been detected from ticks in central Russia near Xinjiang (Shpynov et al., 2004).
Another novel Ehrlichia DNA closely related to E. ewingii was detected in Tibet, Myanmar
and Japan (Inokuma et al., 2004; Parola et al., 2003; Wen et al., 2002). It is possible that
domestic animals in Xinjiang have been infected with some new ehrlichial pathogens and
showed positive antibodies against A. phagocytophilum and E. chaffeensis. Isolation and
characterization of the pathogens will be required for the next step of this study.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science.
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Fig.1. A map of Xinjiang. The three study sites, Altai, Ili and Kashgar, are indicated in the
figure. Urmuqui is the capital city of Xinjiang Uygur Autonomous Region.
Table 1. Information of sera examined from each area
Area Animals Numbers of
Altai Cattle 47
Kashgar Cattle 49
Ili Cattle 50
(Range, years old)
Table 2. Antibody titers against Anaplasma phagocytophilum and Ehrlichia chaffeensis of
domestic animals that showed titers of 1:40 or more against any of the agents
Animals Area No. (years old) A. phagocytophilum
Cattle Altai 12 5
Ili 4 5
Kashgar 34 2
Sheep Altai 10 3-5*
Ili 6 0
Kashgar 8 0
Goats Ili 27 3
Kashgar 8 2
Donkeys Kashgar 50 8
*: Age of individual sheep in Altai was not recorded.
Map of China