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Detection of Anaplasma bovis and Anaplasma phagocytophilum DNA from Haemaphysalis megaspinosa in Hokkaido, Japan
Veterinary Parasitology 168 (2010) 170–172
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Detection of Anaplasma bovis and Anaplasma phagocytophilum DNA from Haemaphysalis megaspinosa in Hokkaido, Japan Kaoru Yoshimoto a, Yuuki Matsuyama a, Hironori Matsuda a, Leo Sakamoto a, Kotaro Matsumoto a, Naoaki Yokoyama b, Hisashi Inokuma a,* a b
Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada, 080-8555, Obihiro, Japan National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 080-8555, Obihiro, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 July 2009 Received in revised form 24 September 2009 Accepted 8 October 2009
DNA from 101 ticks, including Haemaphysalis megaspinosa, Haemaphysalis douglasi, Haemaphysalis flava, Ixodes ovatus and Ixodes persulcatus, collected by flagging in a pasture in Hidaka district (Hokkaido, Japan) was examined for infection with Anaplasma bovis and A. phagocytophilum by species-specific nested PCR. Of the 48 H. megaspinosa nymphs, examined, 1 (2.1%) and 6 (12.5%) were positive for A. bovis and A. phagocytophilum, respectively. One of the 6 positive nymphs was dual positive for both pathogens. Of the 4 larval pools of H. megaspinosa examined, 1 pool was positive for both A. bovis and A. phagocytophilum. None of the samples of the other tick species and adults of H. megaspinosa showed any positive results. These results suggest that H. megaspinosa is a dominant vector tick species of both A. bovis and A. phagocytophilum for cattle in the pasture of Hidaka district, Japan. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anaplasma bovis Anaplasma phagocytophilum Haemaphysalis megaspinosa
1. Introduction Anaplasmosis is a tick-borne infectious disease of cattle, sheep, goats and other domestic ruminants. Several known bacterial species can cause disease in ruminants, including Anaplasma marginale, A. centrale, A. bovis, and A. phagocytophilum (Inokuma, 2007). Until recently, A. marginale and A. centrale were the only reported pathogens of anaplasmosis in Japan (Inokuma et al., 2001; Ooshiro et al., 2009). However, recent molecular studies revealed that cattle in Japan have also been infected by both A. bovis and A. phagocytophilum in Yonaguni island, Okinawa (Ooshiro et al., 2008) and Hidaka district, Hokkaido (Jilintai et al., 2009). In Hokkaido, deer are thought to serve as reservoirs via tick vectors for these Anaplasma spp. (Jilintai et al., 2009). Although DNA fragments of both A. bovis and A. phagocytophilum have been detected from several tick
* Corresponding author. Tel.: +81 155 49 5370; fax: +81 155 49 5370. E-mail address: [email protected] (H. Inokuma). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.008
species, including Ixodes persulcatus, I. ovatus and Haemaphysalis longicornis in Japan (Ohashi et al., 2005; Kawahara et al., 2006; Wuritu et al., 2009), vector ticks of these pathogens for cattle in Hokkaido are still unknown. Therefore, the objective of this study was to determine which tick species may transmit these pathogens to cattle using molecular techniques. 2. Materials and methods 2.1. Tick collection and DNA extraction In June 2008, we collected ticks by flagging at a pasture in Shizunai Livestock Farm, Field Science Center of Northern Biosphere, Hokkaido University. The pastureland is located in Hidaka district, in the south-central part of Hokkaido (428200 N, 1428220 E). This region is surrounded by the Hidaka Mountains to the northeast and the Pacific Ocean to the southwest. Some cattle in this pastureland were infected with A. bovis and A. phagocytophilum without any obvious clinical symptoms (Jilintai et al., 2009). We morphologically identified the collected ticks, and
K. Yoshimoto et al. / Veterinary Parasitology 168 (2010) 170–172
extracted total DNA from each adult or nymphal whole tick using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden) using a previously described method (Hiraoka et al., 2005). Five or six larvae were pooled into one tube and DNA was extracted from each tube. DNA samples were stored at 20 8C in 200 ml of TE buffer until use. 2.2. PCR and sequencing A. bovis and A. phagocytophilum infections were confirmed by 16S rRNA gene-based nested polymerase chain reaction (PCR), as described previously (Kawahara et al., 2006). Briefly, primers EC9 and EC12A were used for primary amplification, and the PCR products were then used as templates for the species-specific second amplification. Primer sets of AB1f and AB1r, and SSAP2f and SSAP2r were used to amplify a 551 bp fragment for A. bovis and a 641 bp fragment for A. phagocytophilum, respectively. For A. phagocytophilum-specific nested PCR, DNA extracted from A. phagocytophilum strain Webster and distilled water were used as positive and negative controls, respectively. No positive controls were used for the A. bovis-specific PCR. To confirm PCR results, PCR product was purified using the Qiaquick PCR purification kit (QIAGEN) for direct sequence analysis using a previously reported method (Inokuma et al., 2007). Nucleotide sequences were confirmed by two additional, independent PCR and sequence analyses. The obtained sequence was compared with other related sequences registered in GenBank. 3. Results and discussion A total of 101 ticks were collected by flagging, including Haemaphysalis megaspinosa (9 females, 2 males, 48 nymphs and 21 larvae), H. douglasi (3 females, 1 male and 1 nymph), H. flava (1 female), Ixodes ovatus (1 female and 7 males) and I. persulcatus (3 females, 1 male and 3 nymphs). In sum, a total of 28 adults and 52 nymphs, and 4 pools of larvae were analyzed to detect A. bovis and A. phagocytophilum by PCR. PCR results are summarized in Table 1. All positive samples were extracted from nymphs or larval pools of H. megaspinosa. Of the 48 nymphs of H. megaspinosa examined, 1 (2.1%) and 6 (12.5%) were positive for A. bovis and A. phagocytophilum, respectively. One of the 6 positive nymphs was dual positive for both pathogens. One of the 4 pools of larval H. megaspinosa was positive for both A. bovis and A. phagocytophilum.
171
In order to confirm the results of the nested PCR, direct sequencing was performed for the positive PCR products. A BLAST search was performed to compare the nucleotide sequences of the PCR products, excluding the primer region (511 bp for A. bovis and 598 bp for A. phagocytophilum), with registered sequences in GenBank. A. bovis-specific-PCR products from a nymph and a pool of larvae of H. megaspinosa were all 100% identical to a strain of A. bovis detected from sika deer in Shimane, Japan (AB211163), sika deer in Korea (EU682764), cattle in China (FJ169957), and cattle in the same pastureland, as previously reported (Jilintai et al., 2009). This strain was also closely related to a strain of A. bovis detected from sika deer in Nara, Japan and H. longicornis in Korea (EU181143), with 99.8% identity (510/511). Hyalomma spp., Rhipicephalus appendiculatus and Amblyomma variegatum are known vectors of A. bovis in African countries (Matson, 1967), but these ticks have not been detected in Japan. A. bovis DNA was also detected from H. longicornis ticks collected from Honshu Island in Japan (Kawahara et al., 2006). The present results suggest that H. megaspinosa is another possible vector tick of A. bovis in Japan. Sequence analysis of A. phagocytophilum-specific positive PCR samples of 6 nymphs and a larval pool of H. megaspinica revealed that all 598 bp of partial 16S rRNA gene sequences were 100% identical to each other and to a registered sequence of A. phagocytophilum detected from sika deer in Hokkaido (AB196720). This sequence was also 99.8% identical to A. phagocytophilum detected from sika deer in Shimane (AB196721) and cattle in Okinawa, Japan (EU368728) with 1 nucleotide difference. It is well known that the main vector of A. phagocytophilum is the I. persulcatus complex, which includes I. persulcatus, I. ricinus, I. pacificus and I. scapularis (Inokuma, 2007). In Japan, A. phagocytophilum DNA was detected from both I. persulcatus and I. ovatus (Ohashi et al., 2005; Wuritu et al., 2009). Given that both I. persulcatus and I. ovatus are common tick species in Hokkaido (Namba, 1963; Yamaguchi et al., 1971), we expected that DNA fragments of A. phagocytophilum would be detected from these tick species. However, none of the samples of the other tick species (H. douglasi, H. flava, I. ovatus and I. persulcatus), and no adult ticks of H. megaspinosa showed any positive results. Because the numbers of these ticks examined were too small in the present study, it is difficult to determine that these negative tick species were not vectors of A. phagocytophilum and A. bovis. However, the present results
Table 1 Detection of DNA fragments of A. bovis and A. phagocytophilum from ticks collected from a pasture in Hokkaido, Japan. Results are given as the number of positive PCR results/number of ticks (larval pools) examined (*). A. bovis
A. phagocytophilum
F
M
N
L
F
M
N
L
H. megaspinosa H. douglasi H. flava I. ovatus I. persulcatus
0/9* 0/3 0/1 0/1 0/3
0/2 0/1 – 0/7 0/1
6/48 0/1 – – 0/3
1/4 – – – –
0/9 0/3 0/1 0/1 0/3
0/2 0/1 – 0/7 0/1
6/48 0/1 – – 0/3
F: female, M: male, N: nymph, L: larva.
1/4 – – – –
172
K. Yoshimoto et al. / Veterinary Parasitology 168 (2010) 170–172
at least suggest that H. megaspinosa is a possible vector tick of A. phagocytophilum in the pasture examined in this area. DNA fragments of A. phagocytophilum were also detected from H. longicornis ticks in Korea (Kim et al., 2003). The Asian strain of A. phagocytophilum may have different biological features from strains found in America or Europe. Furthermore, A. phagocytophilum is thought to be transstadially transmitted by Ixodes ticks and no evidence exists of transovarial transmission (Ogden et al., 1998, 2002; Telford et al., 1996); however, DNA fragment of this pathogen was detected from a larval pool of H. megaspinosa in this study. It is possible that A. phagocytophilum can be transmitted transovarially by H. megaspinosa ticks. Recently, the transovarial transmission of A. phagocytophilum by Dermacentor albipictus was shown in the USA (Baldridge et al., 2009). To demonstrate the transovarial transmission of A. phagocytophilum by ticks, in vitro experiment on laboratory breaded ticks is required. The present study demonstrated that H. megaspinosa may be a vector tick species of both A. bovis and A. phagocytophilum in the pasture of Japan. H. megaspinosa has a wide distribution from Hokkaido, the northern island of Japan, to Kyusyu, the southern island of Japan (Yamaguchi and Kitaoka, 1980). This tick has also been found in a wide range of animals from medium to large sizes, including dogs, cats, deer, wild bores, and bears (Yamaguchi and Kitaoka, 1980; Yamaguchi et al., 1971; Inokuma et al., 2002; Shimada et al., 2003a,b). More epidemiologic studies are needed to determine the distribution of both A. bovis and A. phagocytophilum in animals in Japan. Acknowledgements This study was supported in part by Grant H21Shinkou-Ippan-014 for Research on Emerging and Reemerging Infectious Diseases from the Japanese Ministry of Health, Labor and Welfare, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 18380185 and 21380192). We thank Dr. Hiromi Fujita for assisting with tick identification. References Baldridge, G.D., Scoles, G.A., Burkhardt, N.Y., Schloerder, B., Kurtti, T.J., Munderloh, U.G., 2009. Transovarial transmission of Francisella-like endosymbionts and Anaplasma phagocytophilum variants in Dermacentor albipictus (Acari: Ixodidae). J. Med. Entomol. 46, 625–632. Hiraoka, H., Shimada, Y., Sakata, Y., Watanabe, M., Itamoto, K., Okuda, M., Inokuma, H., 2005. Detection of rickettsial DNA in ixodid ticks recovered from dogs and cats in Japan. J. Vet. Med. Sci. 67, 1217–1222. Inokuma, H., 2007. Vectors and reservoir hosts of Anaplasmataceae. In: Raoult, D., Parola, P. (Eds.), Rickettsial Diseases. Taylor & Grancis Group LLC, New York, pp. 199–212. Inokuma, H., Fujimoto, T., Hosoi, E., Tanaka, S., Fujisaki, K., Okuda, M., Onishi, T., 2002. Tick infestation of sika deer (Cervus nippon) in the
western part of Yamaguchi Prefecture, Japan. J. Vet. Med. Sci. 64, 615– 617. Inokuma, H., Ohashi, M., Jilintai, Tanabe, S., Miyahara, K., 2007. Prevalence of tick-borne rickettsia and Ehrlichia in Ixodes persulcatus and Ixodes ovatus in Tokachi district, Eastern Hokkaido, Japan. J. Vet. Med. Sci. 69, 661–664. Inokuma, H., Terada, Y., Kamio, T., Raoult, D., Brouqui, P., 2001. Analysis of the 16S rRNA gene sequence of Anaplasma centrale and its phylogenetic relatedness to other Ehrlichiae. Clin. Diag. Lab. Immunol. 8, 241– 244. Jilintai, Seino, N., Hayakawa, D., Suzuki, M., Hata, H., Kondo, S., Matsumoto, K., Yokoyama, N., Inokuma, H., 2009. Molecular survey for Anaplasma bovis and Anaplasma phagocytophilum infection in cattle in a pastureland where sika deer appear in Japan. Jpn. J. Infect. Dis. 62, 73–75. Kawahara, M., Rikihisa, Y., Lin, Q., Isogai, E., Tahara, K., Itagaki, A., Hiramitsu, Y., Tajima, T., 2006. Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl. Environ. Microbiol. 72, 1102–1109. Kim, C.-M., Kim, M.-S., Park, M.-S., Park, J.-H., Chae, J.-S., 2003. Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea. Vector-Borne Zoonotic Dis. 3, 17–26. Matson, B.A., 1967. Theileriosis in Rhodesia (Zimbabwe): a study of diagnostic specimens over two seasons. J. S. Afr. Vet. Med. Assoc. 38, 93–97. Namba, N., 1963. Researches on the distribution of the ticks and their habitat in pasture of Hokkaido. Ann. Rep. Hokkaido Agric. Exp. Station 80, 103–114 (in Japanese). Ogden, N.H.K., Bown, K., Horricks, B.K., Woldehiwet, Z., Bennett, M., 1998. Granulocytic Ehrlichia infection in Ixodid ticks and mammals in woodlands and upland of the U.K. Med. Vet. Entomol. 12, 423–429. Ogden, N.H., Casey, A.N.J., French, N.P., Bown, K.J., Adams, J.D.W., Woldehiwet, Z., 2002. Natural Ehrlichia phagocytophia transmission coefficients from sheep ‘carriers’ to Ixodes ricinus ticks vary with the numbers of feeding ticks. Parasitology 124, 127–136. Ohashi, N., Inayoshi, M., Kitamura, K., Kawamori, F., Kawaguchi, D., Nishimura, Y., Naitou, H., Hiroi, M., Masuzawa, T., 2005. Anaplasma phagocytophilum – infected ticks, Japan. Emer. Inf. Dis. 11, 1780– 1782. Ooshiro, M., Zakimi, S., Matsukawa, Y., Katagiri, Y., Inokuma, H., 2008. Detection of Anaplasma bovis and Anaplasma phagocytophilum from cattle on Yonaguni Island, Okinawa, Japan. Vet. Parasitol. 154, 360– 364. Ooshiro, M., Zakimi, S., Matsukawa, Y., Yafuso, M., Katagiri, Y., Inokuma, H., 2009. Anaplasma marginale infection in a Japanese black cow occurred 13 years after eradication of Rhipicephalus (Boophilus) microplus, in Okinawa, Japan. Vet. Parasitol. 160, 351–355. Shimada, Y., Beppu, T., Inokuma, H., Okuda, M., Onishi, T., 2003a. Ixodid tick species recovered from domestic dogs in Japan. Med. Vet. Entomol. 17, 38–45. Shimada, Y., Inokuma, H., Beppu, T., Okuda, M., Onishi, T., 2003b. Survey of ixodid tick species on domestic cat in Japan. Vet. Parasitol. 111, 231– 239. Telford, S.R., Dawson, J.E., Katavlos, P., Warner, C.K., Kolbert, C.P., Persing, D.H., 1996. Perpetuation of the agent of human granulocytic ehrlchiosis in a deer tick–rodent cycle. Proc. Natl. Acad. Sci. U.S.A. 93, 6209–6214. Wuritu, Gaowa, Kawamori, F., Aochi, M., Masuda, T., Ohashi, N., 2009. Characterization of p44/msp2 multigene family of Anaplasma phagocytophilum from two different tick species, Ixodes persulcatus and Ixodes ovatus, in Japan. Jpn. J. Infect. Dis. 62, 142–145. Yamaguchi, N., Kitaoka, S., 1980. Famili Ixodidae. In: Ebara, S. (Ed.), Illustrations of the Mites and Ticks of Japan. Zenkoku Noson Kyoiku Kyokai, Tokyo, (in Japanese), pp. 141–161. Yamaguchi, N., Tipton, V.J., Keegan, H.L., Toshioka, S., 1971. Ticks of Japan, Korea and the Ryukyu Islands. Brigham Young Univ. Sci. Bull. Biol. Ser. 15, 1–226.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Detection of Anaplasma bovis and Anaplasma phagocytophilum DNA from Haemaphysalis megaspinosa in Hokkaido, Japan Kaoru Yoshimoto a, Yuuki Matsuyama a, Hironori Matsuda a, Leo Sakamoto a, Kotaro Matsumoto a, Naoaki Yokoyama b, Hisashi Inokuma a,* a b
Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada, 080-8555, Obihiro, Japan National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 080-8555, Obihiro, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 July 2009 Received in revised form 24 September 2009 Accepted 8 October 2009
DNA from 101 ticks, including Haemaphysalis megaspinosa, Haemaphysalis douglasi, Haemaphysalis flava, Ixodes ovatus and Ixodes persulcatus, collected by flagging in a pasture in Hidaka district (Hokkaido, Japan) was examined for infection with Anaplasma bovis and A. phagocytophilum by species-specific nested PCR. Of the 48 H. megaspinosa nymphs, examined, 1 (2.1%) and 6 (12.5%) were positive for A. bovis and A. phagocytophilum, respectively. One of the 6 positive nymphs was dual positive for both pathogens. Of the 4 larval pools of H. megaspinosa examined, 1 pool was positive for both A. bovis and A. phagocytophilum. None of the samples of the other tick species and adults of H. megaspinosa showed any positive results. These results suggest that H. megaspinosa is a dominant vector tick species of both A. bovis and A. phagocytophilum for cattle in the pasture of Hidaka district, Japan. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anaplasma bovis Anaplasma phagocytophilum Haemaphysalis megaspinosa
1. Introduction Anaplasmosis is a tick-borne infectious disease of cattle, sheep, goats and other domestic ruminants. Several known bacterial species can cause disease in ruminants, including Anaplasma marginale, A. centrale, A. bovis, and A. phagocytophilum (Inokuma, 2007). Until recently, A. marginale and A. centrale were the only reported pathogens of anaplasmosis in Japan (Inokuma et al., 2001; Ooshiro et al., 2009). However, recent molecular studies revealed that cattle in Japan have also been infected by both A. bovis and A. phagocytophilum in Yonaguni island, Okinawa (Ooshiro et al., 2008) and Hidaka district, Hokkaido (Jilintai et al., 2009). In Hokkaido, deer are thought to serve as reservoirs via tick vectors for these Anaplasma spp. (Jilintai et al., 2009). Although DNA fragments of both A. bovis and A. phagocytophilum have been detected from several tick
* Corresponding author. Tel.: +81 155 49 5370; fax: +81 155 49 5370. E-mail address: [email protected] (H. Inokuma). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.008
species, including Ixodes persulcatus, I. ovatus and Haemaphysalis longicornis in Japan (Ohashi et al., 2005; Kawahara et al., 2006; Wuritu et al., 2009), vector ticks of these pathogens for cattle in Hokkaido are still unknown. Therefore, the objective of this study was to determine which tick species may transmit these pathogens to cattle using molecular techniques. 2. Materials and methods 2.1. Tick collection and DNA extraction In June 2008, we collected ticks by flagging at a pasture in Shizunai Livestock Farm, Field Science Center of Northern Biosphere, Hokkaido University. The pastureland is located in Hidaka district, in the south-central part of Hokkaido (428200 N, 1428220 E). This region is surrounded by the Hidaka Mountains to the northeast and the Pacific Ocean to the southwest. Some cattle in this pastureland were infected with A. bovis and A. phagocytophilum without any obvious clinical symptoms (Jilintai et al., 2009). We morphologically identified the collected ticks, and
K. Yoshimoto et al. / Veterinary Parasitology 168 (2010) 170–172
extracted total DNA from each adult or nymphal whole tick using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden) using a previously described method (Hiraoka et al., 2005). Five or six larvae were pooled into one tube and DNA was extracted from each tube. DNA samples were stored at 20 8C in 200 ml of TE buffer until use. 2.2. PCR and sequencing A. bovis and A. phagocytophilum infections were confirmed by 16S rRNA gene-based nested polymerase chain reaction (PCR), as described previously (Kawahara et al., 2006). Briefly, primers EC9 and EC12A were used for primary amplification, and the PCR products were then used as templates for the species-specific second amplification. Primer sets of AB1f and AB1r, and SSAP2f and SSAP2r were used to amplify a 551 bp fragment for A. bovis and a 641 bp fragment for A. phagocytophilum, respectively. For A. phagocytophilum-specific nested PCR, DNA extracted from A. phagocytophilum strain Webster and distilled water were used as positive and negative controls, respectively. No positive controls were used for the A. bovis-specific PCR. To confirm PCR results, PCR product was purified using the Qiaquick PCR purification kit (QIAGEN) for direct sequence analysis using a previously reported method (Inokuma et al., 2007). Nucleotide sequences were confirmed by two additional, independent PCR and sequence analyses. The obtained sequence was compared with other related sequences registered in GenBank. 3. Results and discussion A total of 101 ticks were collected by flagging, including Haemaphysalis megaspinosa (9 females, 2 males, 48 nymphs and 21 larvae), H. douglasi (3 females, 1 male and 1 nymph), H. flava (1 female), Ixodes ovatus (1 female and 7 males) and I. persulcatus (3 females, 1 male and 3 nymphs). In sum, a total of 28 adults and 52 nymphs, and 4 pools of larvae were analyzed to detect A. bovis and A. phagocytophilum by PCR. PCR results are summarized in Table 1. All positive samples were extracted from nymphs or larval pools of H. megaspinosa. Of the 48 nymphs of H. megaspinosa examined, 1 (2.1%) and 6 (12.5%) were positive for A. bovis and A. phagocytophilum, respectively. One of the 6 positive nymphs was dual positive for both pathogens. One of the 4 pools of larval H. megaspinosa was positive for both A. bovis and A. phagocytophilum.
171
In order to confirm the results of the nested PCR, direct sequencing was performed for the positive PCR products. A BLAST search was performed to compare the nucleotide sequences of the PCR products, excluding the primer region (511 bp for A. bovis and 598 bp for A. phagocytophilum), with registered sequences in GenBank. A. bovis-specific-PCR products from a nymph and a pool of larvae of H. megaspinosa were all 100% identical to a strain of A. bovis detected from sika deer in Shimane, Japan (AB211163), sika deer in Korea (EU682764), cattle in China (FJ169957), and cattle in the same pastureland, as previously reported (Jilintai et al., 2009). This strain was also closely related to a strain of A. bovis detected from sika deer in Nara, Japan and H. longicornis in Korea (EU181143), with 99.8% identity (510/511). Hyalomma spp., Rhipicephalus appendiculatus and Amblyomma variegatum are known vectors of A. bovis in African countries (Matson, 1967), but these ticks have not been detected in Japan. A. bovis DNA was also detected from H. longicornis ticks collected from Honshu Island in Japan (Kawahara et al., 2006). The present results suggest that H. megaspinosa is another possible vector tick of A. bovis in Japan. Sequence analysis of A. phagocytophilum-specific positive PCR samples of 6 nymphs and a larval pool of H. megaspinica revealed that all 598 bp of partial 16S rRNA gene sequences were 100% identical to each other and to a registered sequence of A. phagocytophilum detected from sika deer in Hokkaido (AB196720). This sequence was also 99.8% identical to A. phagocytophilum detected from sika deer in Shimane (AB196721) and cattle in Okinawa, Japan (EU368728) with 1 nucleotide difference. It is well known that the main vector of A. phagocytophilum is the I. persulcatus complex, which includes I. persulcatus, I. ricinus, I. pacificus and I. scapularis (Inokuma, 2007). In Japan, A. phagocytophilum DNA was detected from both I. persulcatus and I. ovatus (Ohashi et al., 2005; Wuritu et al., 2009). Given that both I. persulcatus and I. ovatus are common tick species in Hokkaido (Namba, 1963; Yamaguchi et al., 1971), we expected that DNA fragments of A. phagocytophilum would be detected from these tick species. However, none of the samples of the other tick species (H. douglasi, H. flava, I. ovatus and I. persulcatus), and no adult ticks of H. megaspinosa showed any positive results. Because the numbers of these ticks examined were too small in the present study, it is difficult to determine that these negative tick species were not vectors of A. phagocytophilum and A. bovis. However, the present results
Table 1 Detection of DNA fragments of A. bovis and A. phagocytophilum from ticks collected from a pasture in Hokkaido, Japan. Results are given as the number of positive PCR results/number of ticks (larval pools) examined (*). A. bovis
A. phagocytophilum
F
M
N
L
F
M
N
L
H. megaspinosa H. douglasi H. flava I. ovatus I. persulcatus
0/9* 0/3 0/1 0/1 0/3
0/2 0/1 – 0/7 0/1
6/48 0/1 – – 0/3
1/4 – – – –
0/9 0/3 0/1 0/1 0/3
0/2 0/1 – 0/7 0/1
6/48 0/1 – – 0/3
F: female, M: male, N: nymph, L: larva.
1/4 – – – –
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at least suggest that H. megaspinosa is a possible vector tick of A. phagocytophilum in the pasture examined in this area. DNA fragments of A. phagocytophilum were also detected from H. longicornis ticks in Korea (Kim et al., 2003). The Asian strain of A. phagocytophilum may have different biological features from strains found in America or Europe. Furthermore, A. phagocytophilum is thought to be transstadially transmitted by Ixodes ticks and no evidence exists of transovarial transmission (Ogden et al., 1998, 2002; Telford et al., 1996); however, DNA fragment of this pathogen was detected from a larval pool of H. megaspinosa in this study. It is possible that A. phagocytophilum can be transmitted transovarially by H. megaspinosa ticks. Recently, the transovarial transmission of A. phagocytophilum by Dermacentor albipictus was shown in the USA (Baldridge et al., 2009). To demonstrate the transovarial transmission of A. phagocytophilum by ticks, in vitro experiment on laboratory breaded ticks is required. The present study demonstrated that H. megaspinosa may be a vector tick species of both A. bovis and A. phagocytophilum in the pasture of Japan. H. megaspinosa has a wide distribution from Hokkaido, the northern island of Japan, to Kyusyu, the southern island of Japan (Yamaguchi and Kitaoka, 1980). This tick has also been found in a wide range of animals from medium to large sizes, including dogs, cats, deer, wild bores, and bears (Yamaguchi and Kitaoka, 1980; Yamaguchi et al., 1971; Inokuma et al., 2002; Shimada et al., 2003a,b). More epidemiologic studies are needed to determine the distribution of both A. bovis and A. phagocytophilum in animals in Japan. Acknowledgements This study was supported in part by Grant H21Shinkou-Ippan-014 for Research on Emerging and Reemerging Infectious Diseases from the Japanese Ministry of Health, Labor and Welfare, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 18380185 and 21380192). We thank Dr. Hiromi Fujita for assisting with tick identification. References Baldridge, G.D., Scoles, G.A., Burkhardt, N.Y., Schloerder, B., Kurtti, T.J., Munderloh, U.G., 2009. Transovarial transmission of Francisella-like endosymbionts and Anaplasma phagocytophilum variants in Dermacentor albipictus (Acari: Ixodidae). J. Med. Entomol. 46, 625–632. Hiraoka, H., Shimada, Y., Sakata, Y., Watanabe, M., Itamoto, K., Okuda, M., Inokuma, H., 2005. Detection of rickettsial DNA in ixodid ticks recovered from dogs and cats in Japan. J. Vet. Med. Sci. 67, 1217–1222. Inokuma, H., 2007. Vectors and reservoir hosts of Anaplasmataceae. In: Raoult, D., Parola, P. (Eds.), Rickettsial Diseases. Taylor & Grancis Group LLC, New York, pp. 199–212. Inokuma, H., Fujimoto, T., Hosoi, E., Tanaka, S., Fujisaki, K., Okuda, M., Onishi, T., 2002. Tick infestation of sika deer (Cervus nippon) in the
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