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Molecular detection and phylogenetic analysis of Anaplasma bovis from Haemaphysalis longicornis feeding on grazing cattle in Korea
Veterinary Parasitology 196 (2013) 478–481
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Molecular detection and phylogenetic analysis of Anaplasma bovis from Haemaphysalis longicornis feeding on grazing cattle in Korea Huong Thi Thanh Doan a,b,∗,1 , Jin Hyeong Noh a,1 , Se Eun Choe a , Mi Sun Yoo a , Young Ha Kim a , Kondreddy Eswar Reddy a,c , Dong Van Quyen j , Lien Thi Kim Nguyen j , Thuy Thi Dieu Nguyen j , Chang Hee Kweon d , Suk Chan Jung e , Ki Yoon Chang f , Seung Won Kang a,∗∗ a
Parasitology and Insect Disease Research Laboratory, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea Laboratory of Immunology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam c Department of Botany, Plant Molecular Biology Laboratory, S. K. University, Anantapur, Andhrapradesh, India d Systemic Disease Laboratory, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea e Bacterial Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea f Department of Animal and Plant Health Research, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea j Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam b
a r t i c l e
i n f o
Article history: Received 24 December 2012 Received in revised form 12 March 2013 Accepted 21 March 2013
Keywords: Grazing cattle Haemaphysalis longicornis Anaplasma bovis PCR
a b s t r a c t Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNAbased PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4–100%. Two of the four genotypes had high similarity (99.8–100%) with known sequences. The other two genotypes have never been identified. © 2013 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +82 31 467 1824; fax: +82 31 467 1828. ∗∗ Corresponding author. Tel.: +82 31 467 1825. E-mail addresses: [email protected] (H.T.T. Doan), [email protected] (S.W. Kang). 1 Co-first authors (who contributed equally). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.03.025
Anaplasmosis is a tick-borne infectious disease that affects humans and animals throughout the world. These pathogens are transmitted from ticks to their hosts via bites (Sauer et al., 2000). Several species in the genus Anaplasma are known to cause cattle diseases, including A. phagocytophilum, A. centrale, A. bovis, and A. marginale (Inokuma,
H.T.T. Doan et al. / Veterinary Parasitology 196 (2013) 478–481
2007). A. marginale is the main inter-erythrocytic pathogen present in hosts in Japan and China. A. centrale is less pathogenic than A. marginale and has been used as a live vaccine in cattle (De la Fuente et al., 2005). A. phagocytophilum is a causative agent of disease in humans and animals throughout the world, and it has also been detected in a variety of tick and animal species in different areas of Korea. A. bovis is a leukocytotropic agent of bovine anaplasmosis that causes fever, anemia, and weight loss in cattle throughout the tropical and subtropical regions of the world. A. bovis has mainly been detected in Africa, China, and Japan (Ooshiro et al., 2008; Jilintai et al., 2009; Liu et al., 2012), but it was recently found in Korean water deer (Kang et al., 2011) and Haemaphysalis longicornis ticks from Jeju island (Lee and Chae, 2010). However, information about A. bovis is still limited compare with other tick-borne rickettsial pathogens from Korea. In previous studies, H. longicornis was the most common tick species in Korea, where it causes high economic losses in grazing cattle (Lee and Chae, 2010). Therefore, this study investigated the presence of A. bovis infections in H. longicornis ticks sampled from grazing cattle in various areas of Korea. The present study also analyzed the genotypes of A. bovis present in H. longicornis ticks from Korea. 2. Materials and methods During 2010–2011, 865 adult H. longicornis ticks were collected from grazing cattle in the five Korean provinces (Jeju, Gyeongbuk, Chungbuk, Jeonbuk, and Jeonnam) based on microscopic examinations (Yamaguti et al., 1971) (Fig. 1). Subsequently, according to sampling locations ticks were pooled into 535 samples (1–6 ticks per samples pool) (Fig. 1) and stored at −20 ◦ C until use. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany), according to the manufacturer’s instructions. Infection with A. bovis was determined based on the presence of the 16S rRNA gene in ticks, and speciesspecific PCR reactions were conducted using the specific primer pair (AB1f/AB1r) and PCR conditions (heating at 94 ◦ C for 5 min; followed by 40 cycles of denaturation at 94 ◦ C for 1 min, annealing at 55 ◦ C for 1 min, and extension at 72 ◦ C for 1 min) reported by Kawahara et al. (2006). Negative controls and positive controls were included in each PCR experiment. The PCR products were then separated by 1.5% agarose gels electrophoresis, stained with ethidium bromide, and photographed under UV light. The expected amplicon size was about 551 bp. The strong bands of approximately 551 bp determined by positive amplification were purified using a Qiagen Gel Extraction Kit (Qiagen, Germany) and used directly for sequencing (Macrogen, Korea). The nucleotide sequences were identified using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) database website (http://www.ncbi.nlm.nih.gov/BLAST). All sequences shown in this study had the primer region sequence removed before analysis. A multiple nucleotide alignment was produced using published A. bovis sequences according to GENEDOC version 2.5 (Nicholas et al., 1997). A phylogenetic tree was constructed using MEGA 4.0 (Tamura et al., 2007) with the
479
GW GG
25/ 151 (55/ 4 )
CN
CB
GB 12/ 82 (25/ 2 )
JB 3/ 32 (32/ 1 )
GN 0/ 198 (80/ 3)
JN
J 10/ 72 (40/ 2 )
Fig. 1. Map of Republic of Korea indicating the five provinces where specimens were collected (marked as solid square). The Korean provinces are as follows: Gyeonggi (GG); Gangwon (GW); Chungnam (CN); Chungbuk (CB); Jeonbuk (JB); Gyeongbuk (GB); Jeonnam (JN); Gyeongnam (GN); Jeju (J). Underlined numerals indicate the numbers of positive and tested samples, respectively. Numerals in parentheses indicate the numbers of tested cattle and herds, respectively.
neighbor-joining method. The phylogenies were tested with 1000 bootstrap replicates. The A. bovis sequences obtained in this study were deposited in GenBank under the following accession numbers: KC311344, KC311345, KC311346, and KC311347. The GenBank accession numbers of the 16S rRNA gene sequences used to construct the phylogenetic trees are shown in Fig. 2.
3. Results and discussion H. longicornis is the most common tick species in Korea and is known to be a vector of many important pathogens that affect animals (Kim et al., 2006; Lee and Chae, 2010). In Korea, A. bovis infection was detected the first time from H. longicornis feeding on Hedgehog in Gyeonggi province. However, reports of this pathogen are limited. Present study was performed to investigate the presence of A. bovis in this ticks feeding on cattle in five provinces of South Korea. The 16S rRNA gene is considered to be a sensitive molecular tool for the detection of Anaplasma species and in phylogenic studies (Kang et al., 2011), so it was used in this study. A. bovis was detected in 50/535 samples (9.3%), i.e., 10/72 (14%) from Jeju, 25/151 (16.5%) from Chungbuk, 12/82 (14.6%) from Gyeongbuk, and 3/32 (9.3%) from Jeonbuk. However, infections were not found at the sites in
480
H.T.T. Doan et al. / Veterinary Parasitology 196 (2013) 478–481
A.bovis from ruminant in Africa (U03775) A.bovis from H. concinna tick in Russia (JX092094) A.bovis T-KOAB2 (KC311345) A.bovis from deer in Japan (AB588965) 64 A.bovis from deer in Korea (EU181142) A.bovis from Tick in Japan (AB196475) 51 A. bovis T-KOAB3 (KC311346) A. bovis T-KOAB4 (KC311347) 52 A.bovis from deer in Korea (EU682764) A. bovis T-KOAB1 (KC311344) 100 A.bovis from goat in China (JN558829) A.bovis from H. longicornis tick in Korea (GU064902) A.platys from dog in Germany (JQ396431) A.platys from dog in Malaysia (JF683610) A.platys from dog in Thailand (EF139459) A.platys from dog in France (AF303467) A.phagocytophilum from H. longicornis tick in Korea (GU064899) A.phagocytophilum from horse in Sweden (AY527213) A.phagocytophilum from tick in Japan (AY969015) 99 A.phagocytophilum from tick in Estonia (HQ629917) A.phagocytophilum Germany (JX173652) A.centraleFrance(AF283007) 67 A.centrale from cattle in Italy (EF520686) 97 A.centrale from tick in Netherlands (AF318944) A.marginale from ruminant in China (HM538192) 50 A.marginale USA (AF311303) 42 A.marginale Australia (AF414874) A.marginale from cattle in Philippine (JQ839012) 65
97
52
0.002
Fig. 2. Phylogenetic relationships of A. bovis isolates from H. longicornis ticks in Korea (bold letters) and known Anaplasma species based on the nucleotide sequences of the partial 511 bp 16S rRNA gene. The phylogenetic tree was produced using MEGA 4.0 (Tamura et al., 2007) with bootstrap values of 1000 replicates and the neighbor-joining method.
Jeonnam, although this location had a high rate of tickborne rickettsial pathogens (72%, data not shown). Fifty positive PCR products were sequenced successfully and these confirmed the PCR results. A BLAST search and GENEDOC version 2.5 was performed to compare the nucleotide sequences of the PCR products, excluding the primer region (511 bp), with sequences in GenBank. The 50 Korean isolates had similarities of 99.4–100% and they formed four clusters in the phylogenetic tree based on their sequence similarity: T-KOAB1 (n = 28), T-KOAB2 (n = 9), TKOAB3 (n = 5), and T-KOAB4 (n = 8) (Fig. 2). Twenty-eight Korean isolates with the T-KOAB1 genotype had close relationships with known sequences from a Korean H. longicornis tick (GenBank accession number GU064902), a Korean deer (GenBank accession number EU682764), and a Chinese goat (GenBank accession number HQ913644, JN558829) (99.9–100% nucleotide similarity). Nine Korean isolates with the T-KOAB2 genotype had close relationships with sequences from a Korean deer (GenBank accession number EU181142), a Japanese deer (GenBank accession number AB588965), and a Japanese tick (GenBank accession number AB196475) (99.9–100% nucleotide similarity). Thirteen isolates belonged to two new independent genotypes (T-KOAB3 and T-KOAB4), that the sequences have
never been found previously. The isolates in each of the two new A. bovis genotypes shared 100% similarity. These sequences was 99.6% and 99.4% identical to the sequence from Korean deer (GenBank accession number EU181142), respectively. The similarity between the four genotypes was 99.4–99.7%. Geographic segregation of A. bovis isolates was not detected in this study (Table 1). A. bovis was first described in 1936 in Iran (Donatien and Lestoquard, 1936). It has been detected in many countries since then and it is most common in domestic cattle from Iran, Africa, Brazil, China, and Japan (Noaman and Shayan,
Table 1 Number of A. bovis genotypes identified in H. longicornis feeding in grazing cattle in five Korean provinces during 2010–2011. Regions
n
No. of A. bovis genotypes T-KOAB1 T-KOAB2 T-KOAB3 T-KOAB4 Total
Gyeongbuk 82 Chungbuk 151 Jeonbuk 32 Jeonnam 198 Jeju 72
5 17 1 0 5
2 3 1 0 3
1 2 1 0 1
4 3 0 0 1
12 25 3 0 10
Total
28
9
5
8
50
535
H.T.T. Doan et al. / Veterinary Parasitology 196 (2013) 478–481
2010; Kawahara et al., 2006). In Japan, A. bovis has been isolated from cattle (15–53.3%), deer (23%) (Jilintai et al., 2009; Ooshiro et al., 2008), and H. longicornis tick (12%) (Kawahara et al., 2006). A. bovis has been isolated from goats in China (49.6%) (Liu et al., 2012). This infection was present in 34.8% of water deer (Kang et al., 2011) in Korea, and it was also detected in H. longicornis ticks from Jeju and Gyeonggi after the sequencing of positive PCR results for A. phagocytophilum and subsequent analysis using BLAST at NCBI (Lee and Chae, 2010; Kim et al., 2003). To the best of our knowledge, this is the first report to demonstrate the presence of A. bovis in ticks from the Chungbuk, Jeonbuk, and Geonbuk provinces of Korea. The detection of different A. bovis genotypes in this study as well as the presence of this agent in various areas suggests that Korea may be a geographic reservoir for A. bovis infection. The presence of A. bovis in ticks feeding on cattle shows that these cattle may be at risk of infection. Moreover, A. bovis can persist for a long time in infected animals (Liu et al., 2012). In conclusion, the present study detected A. bovis infection from H. longicornis feeding on grazing cattle in various new areas of Korea and four genotypes were identified. Further studies of cattle, other animals, and vector-borne diseases will be necessary in these parts of Korea. Acknowledgment This study was supported by the Animal, Plant, and Fisheries Quarantine and Inspection Agency, Korean Ministry of Agriculture and Forestry, Republic of Korea. References De la Fuente, J., Lew, A., Lutz, H., Meli, M.L., Hofmann-Lehmann, R., Shkap, V., Molad, T., Mangold, A.J., Almazán, C., Naranjo, V., Gortázar, C., Torina, A., Caracappa, S., García-Pérez, A.L., Barral, M., Oporto, B., Ceci, L., Carelli, G., Blouin, E.F., Kocan, K.M., 2005. Genetic diversity of Anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development. Anim. Health Res. Rev. 6, 75–89.
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Donatien, A., Lestoquard, F., 1936. Rickettsia bovis, novella espece pathogen pour le boeuf. Bull. Soc. Pathol. Exot. 29, 1057–1061. 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. Jilintai, N.S., 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 Hokkaido, Japan. Jpn. J. Infect. Dis. 62 (1), 73–75. Kang, J.G., Ko, S., Kim, Y.J., Yang, H.J., Lee, H., Shin, N.S., Choi, K.S., Chae, J.S., 2011. New genetic variants of Anaplasma phagocytophilum and Anaplasma bovis from Korean Water Deer (Hydropotes inermis argyropus). Vector Borne Zoonot. Dis. 11 (7), 929–938. Kawahara, M., Rikihisa, Y., Lin, Q., Isogai, E., Tahara, K., Itagaki, A., Hiramitshu, 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 (2), 1102–1109. Kim, C.M., Kim, M.S., Park, M.S., Park, J.H., Chae, J.S., 2003. Identification of Ehrlichia chaffensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea. Vector Borne Zoonot. Dis. 3 (1), 17–26. Kim, C.M., Yi, Y.H., Yu, D.H., Lee, M.J., Cho, M.R., Desai, A.R., Shringi, S., Klein, T.A., Kim, H.C., Song, J.W., Baek, L.J., Chong, S.T., Guinn, M.L.O., Lee, J.S., Lee, I.Y., Park, J.H., Foley, J., Chae, J.S., 2006. Tick-borne rickettsial pathogens in ticks and small mammals in Korea. Appl. Environ. Microbiol. 72 (9), 5766–5776. Lee, M.J., Chae, J.S., 2010. Molecular detection of Ehrlichia chaffeensis and Anaplasma bovis in the salivary glands from Haemaphysalis longicornis ticks. Vector Borne Zoonot. Dis. 10 (4), 411–413. Liu, Z., Ma, M., Wang, Z., Wang, J., Peng, Y., Li, Y., Guan, G., Luo, J., Yin, H., 2012. Molecular survey and genetic identification of Anaplasma species in goats from central and southern China. Appl. Environ. Microbiol. 78 (2), 464–470. Nicholas, K.B., Nicholas, H.B., Deerfield, D.W., 1997. GeneDoc: analysis and visualization of genetic variation. EMBNEW. NEWS 4, 14. Noaman, V., Shayan, P., 2010. Molecular detection of Anaplasma bovis in cattle from central part of Iran. Vet Res. Forum 1, 117–122. 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. Sauer, J.R., Essenberg, R.C., Bowman, A.S., 2000. Salivary glands in ixodid ticks: control and mechanism of secretion. J. Insect Physiol. 46, 1069–1078. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Yamaguti, N., Tipton, V.J., Keegan, H., Toshioka, S., 1971. Ticks of Japan, Korea, and the Ryukyu islands. Brigham Young Univ. Sci. Bull. Biol. Ser. 15, 1–126.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Molecular detection and phylogenetic analysis of Anaplasma bovis from Haemaphysalis longicornis feeding on grazing cattle in Korea Huong Thi Thanh Doan a,b,∗,1 , Jin Hyeong Noh a,1 , Se Eun Choe a , Mi Sun Yoo a , Young Ha Kim a , Kondreddy Eswar Reddy a,c , Dong Van Quyen j , Lien Thi Kim Nguyen j , Thuy Thi Dieu Nguyen j , Chang Hee Kweon d , Suk Chan Jung e , Ki Yoon Chang f , Seung Won Kang a,∗∗ a
Parasitology and Insect Disease Research Laboratory, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea Laboratory of Immunology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam c Department of Botany, Plant Molecular Biology Laboratory, S. K. University, Anantapur, Andhrapradesh, India d Systemic Disease Laboratory, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea e Bacterial Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea f Department of Animal and Plant Health Research, Animal, Plant and Fisheries Quarantine and Inspection Agency, 480 Anyang 6 dong, Anyang 420-480, Republic of Korea j Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam b
a r t i c l e
i n f o
Article history: Received 24 December 2012 Received in revised form 12 March 2013 Accepted 21 March 2013
Keywords: Grazing cattle Haemaphysalis longicornis Anaplasma bovis PCR
a b s t r a c t Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNAbased PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4–100%. Two of the four genotypes had high similarity (99.8–100%) with known sequences. The other two genotypes have never been identified. © 2013 Elsevier B.V. All rights reserved.
1. Introduction
∗ Corresponding author. Tel.: +82 31 467 1824; fax: +82 31 467 1828. ∗∗ Corresponding author. Tel.: +82 31 467 1825. E-mail addresses: [email protected] (H.T.T. Doan), [email protected] (S.W. Kang). 1 Co-first authors (who contributed equally). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.03.025
Anaplasmosis is a tick-borne infectious disease that affects humans and animals throughout the world. These pathogens are transmitted from ticks to their hosts via bites (Sauer et al., 2000). Several species in the genus Anaplasma are known to cause cattle diseases, including A. phagocytophilum, A. centrale, A. bovis, and A. marginale (Inokuma,
H.T.T. Doan et al. / Veterinary Parasitology 196 (2013) 478–481
2007). A. marginale is the main inter-erythrocytic pathogen present in hosts in Japan and China. A. centrale is less pathogenic than A. marginale and has been used as a live vaccine in cattle (De la Fuente et al., 2005). A. phagocytophilum is a causative agent of disease in humans and animals throughout the world, and it has also been detected in a variety of tick and animal species in different areas of Korea. A. bovis is a leukocytotropic agent of bovine anaplasmosis that causes fever, anemia, and weight loss in cattle throughout the tropical and subtropical regions of the world. A. bovis has mainly been detected in Africa, China, and Japan (Ooshiro et al., 2008; Jilintai et al., 2009; Liu et al., 2012), but it was recently found in Korean water deer (Kang et al., 2011) and Haemaphysalis longicornis ticks from Jeju island (Lee and Chae, 2010). However, information about A. bovis is still limited compare with other tick-borne rickettsial pathogens from Korea. In previous studies, H. longicornis was the most common tick species in Korea, where it causes high economic losses in grazing cattle (Lee and Chae, 2010). Therefore, this study investigated the presence of A. bovis infections in H. longicornis ticks sampled from grazing cattle in various areas of Korea. The present study also analyzed the genotypes of A. bovis present in H. longicornis ticks from Korea. 2. Materials and methods During 2010–2011, 865 adult H. longicornis ticks were collected from grazing cattle in the five Korean provinces (Jeju, Gyeongbuk, Chungbuk, Jeonbuk, and Jeonnam) based on microscopic examinations (Yamaguti et al., 1971) (Fig. 1). Subsequently, according to sampling locations ticks were pooled into 535 samples (1–6 ticks per samples pool) (Fig. 1) and stored at −20 ◦ C until use. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germany), according to the manufacturer’s instructions. Infection with A. bovis was determined based on the presence of the 16S rRNA gene in ticks, and speciesspecific PCR reactions were conducted using the specific primer pair (AB1f/AB1r) and PCR conditions (heating at 94 ◦ C for 5 min; followed by 40 cycles of denaturation at 94 ◦ C for 1 min, annealing at 55 ◦ C for 1 min, and extension at 72 ◦ C for 1 min) reported by Kawahara et al. (2006). Negative controls and positive controls were included in each PCR experiment. The PCR products were then separated by 1.5% agarose gels electrophoresis, stained with ethidium bromide, and photographed under UV light. The expected amplicon size was about 551 bp. The strong bands of approximately 551 bp determined by positive amplification were purified using a Qiagen Gel Extraction Kit (Qiagen, Germany) and used directly for sequencing (Macrogen, Korea). The nucleotide sequences were identified using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) database website (http://www.ncbi.nlm.nih.gov/BLAST). All sequences shown in this study had the primer region sequence removed before analysis. A multiple nucleotide alignment was produced using published A. bovis sequences according to GENEDOC version 2.5 (Nicholas et al., 1997). A phylogenetic tree was constructed using MEGA 4.0 (Tamura et al., 2007) with the
479
GW GG
25/ 151 (55/ 4 )
CN
CB
GB 12/ 82 (25/ 2 )
JB 3/ 32 (32/ 1 )
GN 0/ 198 (80/ 3)
JN
J 10/ 72 (40/ 2 )
Fig. 1. Map of Republic of Korea indicating the five provinces where specimens were collected (marked as solid square). The Korean provinces are as follows: Gyeonggi (GG); Gangwon (GW); Chungnam (CN); Chungbuk (CB); Jeonbuk (JB); Gyeongbuk (GB); Jeonnam (JN); Gyeongnam (GN); Jeju (J). Underlined numerals indicate the numbers of positive and tested samples, respectively. Numerals in parentheses indicate the numbers of tested cattle and herds, respectively.
neighbor-joining method. The phylogenies were tested with 1000 bootstrap replicates. The A. bovis sequences obtained in this study were deposited in GenBank under the following accession numbers: KC311344, KC311345, KC311346, and KC311347. The GenBank accession numbers of the 16S rRNA gene sequences used to construct the phylogenetic trees are shown in Fig. 2.
3. Results and discussion H. longicornis is the most common tick species in Korea and is known to be a vector of many important pathogens that affect animals (Kim et al., 2006; Lee and Chae, 2010). In Korea, A. bovis infection was detected the first time from H. longicornis feeding on Hedgehog in Gyeonggi province. However, reports of this pathogen are limited. Present study was performed to investigate the presence of A. bovis in this ticks feeding on cattle in five provinces of South Korea. The 16S rRNA gene is considered to be a sensitive molecular tool for the detection of Anaplasma species and in phylogenic studies (Kang et al., 2011), so it was used in this study. A. bovis was detected in 50/535 samples (9.3%), i.e., 10/72 (14%) from Jeju, 25/151 (16.5%) from Chungbuk, 12/82 (14.6%) from Gyeongbuk, and 3/32 (9.3%) from Jeonbuk. However, infections were not found at the sites in
480
H.T.T. Doan et al. / Veterinary Parasitology 196 (2013) 478–481
A.bovis from ruminant in Africa (U03775) A.bovis from H. concinna tick in Russia (JX092094) A.bovis T-KOAB2 (KC311345) A.bovis from deer in Japan (AB588965) 64 A.bovis from deer in Korea (EU181142) A.bovis from Tick in Japan (AB196475) 51 A. bovis T-KOAB3 (KC311346) A. bovis T-KOAB4 (KC311347) 52 A.bovis from deer in Korea (EU682764) A. bovis T-KOAB1 (KC311344) 100 A.bovis from goat in China (JN558829) A.bovis from H. longicornis tick in Korea (GU064902) A.platys from dog in Germany (JQ396431) A.platys from dog in Malaysia (JF683610) A.platys from dog in Thailand (EF139459) A.platys from dog in France (AF303467) A.phagocytophilum from H. longicornis tick in Korea (GU064899) A.phagocytophilum from horse in Sweden (AY527213) A.phagocytophilum from tick in Japan (AY969015) 99 A.phagocytophilum from tick in Estonia (HQ629917) A.phagocytophilum Germany (JX173652) A.centraleFrance(AF283007) 67 A.centrale from cattle in Italy (EF520686) 97 A.centrale from tick in Netherlands (AF318944) A.marginale from ruminant in China (HM538192) 50 A.marginale USA (AF311303) 42 A.marginale Australia (AF414874) A.marginale from cattle in Philippine (JQ839012) 65
97
52
0.002
Fig. 2. Phylogenetic relationships of A. bovis isolates from H. longicornis ticks in Korea (bold letters) and known Anaplasma species based on the nucleotide sequences of the partial 511 bp 16S rRNA gene. The phylogenetic tree was produced using MEGA 4.0 (Tamura et al., 2007) with bootstrap values of 1000 replicates and the neighbor-joining method.
Jeonnam, although this location had a high rate of tickborne rickettsial pathogens (72%, data not shown). Fifty positive PCR products were sequenced successfully and these confirmed the PCR results. A BLAST search and GENEDOC version 2.5 was performed to compare the nucleotide sequences of the PCR products, excluding the primer region (511 bp), with sequences in GenBank. The 50 Korean isolates had similarities of 99.4–100% and they formed four clusters in the phylogenetic tree based on their sequence similarity: T-KOAB1 (n = 28), T-KOAB2 (n = 9), TKOAB3 (n = 5), and T-KOAB4 (n = 8) (Fig. 2). Twenty-eight Korean isolates with the T-KOAB1 genotype had close relationships with known sequences from a Korean H. longicornis tick (GenBank accession number GU064902), a Korean deer (GenBank accession number EU682764), and a Chinese goat (GenBank accession number HQ913644, JN558829) (99.9–100% nucleotide similarity). Nine Korean isolates with the T-KOAB2 genotype had close relationships with sequences from a Korean deer (GenBank accession number EU181142), a Japanese deer (GenBank accession number AB588965), and a Japanese tick (GenBank accession number AB196475) (99.9–100% nucleotide similarity). Thirteen isolates belonged to two new independent genotypes (T-KOAB3 and T-KOAB4), that the sequences have
never been found previously. The isolates in each of the two new A. bovis genotypes shared 100% similarity. These sequences was 99.6% and 99.4% identical to the sequence from Korean deer (GenBank accession number EU181142), respectively. The similarity between the four genotypes was 99.4–99.7%. Geographic segregation of A. bovis isolates was not detected in this study (Table 1). A. bovis was first described in 1936 in Iran (Donatien and Lestoquard, 1936). It has been detected in many countries since then and it is most common in domestic cattle from Iran, Africa, Brazil, China, and Japan (Noaman and Shayan,
Table 1 Number of A. bovis genotypes identified in H. longicornis feeding in grazing cattle in five Korean provinces during 2010–2011. Regions
n
No. of A. bovis genotypes T-KOAB1 T-KOAB2 T-KOAB3 T-KOAB4 Total
Gyeongbuk 82 Chungbuk 151 Jeonbuk 32 Jeonnam 198 Jeju 72
5 17 1 0 5
2 3 1 0 3
1 2 1 0 1
4 3 0 0 1
12 25 3 0 10
Total
28
9
5
8
50
535
H.T.T. Doan et al. / Veterinary Parasitology 196 (2013) 478–481
2010; Kawahara et al., 2006). In Japan, A. bovis has been isolated from cattle (15–53.3%), deer (23%) (Jilintai et al., 2009; Ooshiro et al., 2008), and H. longicornis tick (12%) (Kawahara et al., 2006). A. bovis has been isolated from goats in China (49.6%) (Liu et al., 2012). This infection was present in 34.8% of water deer (Kang et al., 2011) in Korea, and it was also detected in H. longicornis ticks from Jeju and Gyeonggi after the sequencing of positive PCR results for A. phagocytophilum and subsequent analysis using BLAST at NCBI (Lee and Chae, 2010; Kim et al., 2003). To the best of our knowledge, this is the first report to demonstrate the presence of A. bovis in ticks from the Chungbuk, Jeonbuk, and Geonbuk provinces of Korea. The detection of different A. bovis genotypes in this study as well as the presence of this agent in various areas suggests that Korea may be a geographic reservoir for A. bovis infection. The presence of A. bovis in ticks feeding on cattle shows that these cattle may be at risk of infection. Moreover, A. bovis can persist for a long time in infected animals (Liu et al., 2012). In conclusion, the present study detected A. bovis infection from H. longicornis feeding on grazing cattle in various new areas of Korea and four genotypes were identified. Further studies of cattle, other animals, and vector-borne diseases will be necessary in these parts of Korea. Acknowledgment This study was supported by the Animal, Plant, and Fisheries Quarantine and Inspection Agency, Korean Ministry of Agriculture and Forestry, Republic of Korea. References De la Fuente, J., Lew, A., Lutz, H., Meli, M.L., Hofmann-Lehmann, R., Shkap, V., Molad, T., Mangold, A.J., Almazán, C., Naranjo, V., Gortázar, C., Torina, A., Caracappa, S., García-Pérez, A.L., Barral, M., Oporto, B., Ceci, L., Carelli, G., Blouin, E.F., Kocan, K.M., 2005. Genetic diversity of Anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development. Anim. Health Res. Rev. 6, 75–89.
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