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ORIGINAL ARTICLE
Molecular Detection of Anaplasma, Bartonella,and Borrelia Species in Ticks Collected from Migratory
Birds from Hong-do Island, Republic of Korea
Jun-Gu Kang,1 Heung-Chul Kim,2 Chang-Yong Choi,3 Hyun-Young Nam,3 Hee-Young Chae,3
Sung-Tae Chong,2 Terry A. Klein,4 Sungjin Ko,1 and Joon-Seok Chae1
Abstract
Bird migration is a recurring annual and seasonal event undertaken by more than 100 species of birds in thesoutheast Asian and northeast Palearctic regions that pass through or remain for short periods from April toMay and September to November at Hong-do Island, Republic of Korea (ROK). A total of 212 ticks (40 Hae-maphysalis flava, 12 H. longicornis, 146 Ixodes turdus, 13 I. nipponensis, and 1 I. ornithophila) were collected from 65/2,161 (3.0%) migratory birds consisting of 21 species that were captured from January, 2008, through December,2009, as part of the Migratory Birds Center, Hong-do bird banding program for studying bird migration patterns.Adult ticks were assayed individually while larvae and nymphs were pooled (1–22 and 1–6 ticks per pool,respectively) into 31 and 65 pools, respectively. Ticks were assayed for zoonotic pathogens by PCR using 16SrRNA, heat shock protein (groEL), and internal transcribed spacer (ITS) gene primers to amplify genera specific forAnapalsma, Bartonella, and Borrelia PCR amplicons. Using the 16S rRNA-based nested PCR, A. phagocytophilum(n = 1) was detected in I. nipponensis collected from Zoothera sibirica and A. bovis (n = 1) was detected in I. turduscollected from Emberiza chrysophrys. Borrelia turdi 16S rRNA genes (n = 3) were detected in I. turdus and I. nippo-nensis collected from Turdus pallidus and Zoothera aurea. Borrelia spp. 16S rRNA genes (n = 4) were detected in Ixodesticks collected from Emberiza tristrami, T. pallidus, and Z. aurea. The Bartonella grahamii ITS gene (n = 1) was detectedby nested PCR assay in I. turdus collected from Z. aurea. These results provide insight into the potential role ofmigratory birds in the dispersal of ticks and associated tick-borne pathogens throughout their ranges in Asia.
Key Words: Anaplasma—Bartonella—Borrelia—Tick-borne pathogens—Migratory bird—Korea.
Introduction
Migratory birds transport ectoparasites, includingticks, and associated pathogens on their migratory
routes between breeding and nonbreeding grounds. There-fore, this may result in the introduction of exotic tick speciesand associated tick-borne pathogens over long distances anddiverse environments. It is well known that migratory birdsmay play an important role in the long distance dispersal ofBorrelia burgdorferi, the causative agent of Lyme disease(Miyamoto et al. 1993, Smith et al. 1996, Miyamoto et al. 1997),but there are limited studies available on the role of migratorybirds in the transmission of other tick-borne pathogens,
i.e., Anaplasma (Ogden et al. 2008, Paulauskas et al. 2009,Hildebrandt et al. 2010, Palomar et al. 2012) and Bartonellaspecies.
Anaplasma phagocytophilum, the causative agent of humangranulocytic anaplasmosis (HGA), transmitted primarily byIxodes spp. ticks, has a northern hemisphere distribution(Chen et al. 1994). Recently in the Republic of Korea (ROK),A. phagocytophilum was detected by PCR technologies in deerand small mammals and in Haemaphysalis and Ixodes spp.collected by tick drag (Kim et al. 2006, Oh et al. 2009, Kanget al. 2011). Although Anaplasma bovis has been primarilydetected in African cattle, it also was detected in Ixodes andHaemaphysalis spp. ticks, and deer in Japan and the ROK
1Laboratory of Veterinary Internal Medicine, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul NationalUniversity, Seoul, Korea.
25th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, Unit 15247, APO AP96205-5247, USA.3Migratory Birds Center, National Park Research Institute, Heuksan-myeon, Shinan-gun, Jeollanam Province, Korea.4Force Health Protection and Preventive Medicine, 65th Medical Brigade/US Army MEDDAC-Korea, Unit 15281, APO AP 96205-5281, USA.
VECTOR-BORNE AND ZOONOTIC DISEASESVolume 13, Number X, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2012.1149
1
(Kawahara et al. 2006, Kim et al. 2006, Lee et al. 2009, Oh et al.2009, Kang et al. 2011).
Bartonella grahamii, the zoonotic agent causing neuror-etinitis, was first identified from a rodent in the UnitedKingdom (Birtles et al. 1994, Kerkhoff et al. 1999). In Asia,B. grahamii is the predominant species detected from rodentsin neighboring countries, i.e., China (Liu et al. 2010) and Japan(Inoue et al. 2009, Kabeya et al. 2011).
Borrelia turdi was firstly characterized and isolated fromIxodes turdus, a less commonly collected species from Japan(Fukunaga et al. 1996), and more recently from Ixodes ricinuscollected from passerine birds from Norway (Hasle et al.2011). Both papers indicated that the host of B. turdi was birds,but the pathogenesis of B. turdi is not known and reports havebeen rare.
The purpose of this investigation was to determine theprevalence of Anaplasma, Bartonella, and Borrelia species inticks collected from migratory birds that are potential hostsfor the introduction of exotic tick species and reservoirs for thedispersal of the pathogens throughout their migratory routesin southeast Asia.
Materials and Methods
Survey area
The Migratory Birds Center of the National Park ResearchInstitute, located on Hong-do Island (34�41’07’’N,125�11’33’’E), conducted bird banding surveys from January,2008, to December, 2009, to understand migration routes and
strategies of migratory birds (Fig. 1). Hong-do Island, desig-nated as National Monument No. 170 (April, 1965), is a small(6.4 km2) protected remote southwestern island located inHeuksan-myeon (district), Shinan-gun (county), Jeollanam-do (province), and 115 km west of Mokpo (mainland portcity). Migratory birds pass through Hong-do and the ROKmainland during the spring and autumn migration alongtheir south and northeast Asian flyways.
Bird and tick collections
Migratory birds were captured on Hong-do using two 36-mm-meshed mist nets (12 meters in length, 2.5 meters high)placed at the ground level. The trapped birds were identifiedto species, banded with an identification number, and theirsexes and ages determined. Banded birds were examinedclosely, particularly around the head and neck, for ticks andother ectoparasites prior to release. Ticks were secured bytheir mouthparts next to the skin with a fine forceps andgently removed to prevent injury to the bird. The removedticks were placed individually in cryovials containing 70%ethyl alcohol and labeled with a unique identification numberthat corresponded to the migratory bird collection data. Theticks were identified microscopically to stage of developmentand species using taxonomic identification keys (Yamagutiet al. 1971, Hoogstraal and Wassef 1973). An electronic datasheet, which included the unique bird identification number,species, sex, and other pertinent information, was completed,and the ticks were assayed by PCR techniques for rickettsialand bacterial pathogens.
FIG. 1. Hong-do Island, the collection site of ticks removed from migratory during the Migratory Birds Center bird bandingsurveys, Jeollanam-do Province, Republic of Korea.
2 KANG ET AL.
Table 1. Nucleotide Sequences Of PCR Primers and Conditions for Amplification
of Anaplasma, Bartonella, and Borrelia Species Genes
Primer sequences (5¢- 3¢)
Species andtarget genes
Name of PCRprimers andconditions
Denaturation(oC/min)
Annealing(oC/min)
Extension(oC/min) Cycles
PCRproductssize (bp) References
Anaplasma andEhrlichia spp.16S rRNA
AE1-F AAGCTTAACACATGCAAGTCGAA 1,406 Oh et al. (2009)AE1-R AGTCACTGACCCAACCTTAAATGConditions 94/1 56/1 72/1.5 35
A. phagocytophilum16S rRNA
EE3 GTCGAACGGATTATTCTTTATAGCTTGC 926 Barlough et al. (1996)EE4 CCCTTCCGTTAAGAAGGATCTAATCTCCConditions 94/0.5 56/0.5 72/0.75 25
A. phagocytophilumgroEL
HS1a AITGGGCTGGTAITGAAT 1,433 Liz et al. (2000)HS6a CCICCIGGIACIAIACCTTCConditions 88/1 48/2 68/1.5 40HS43 ATWGCWAARGAAGCATAGTC 1,297 Lotric-Furlan et al. (1998)HSVR CTCAACAGCAGCTCTAGTAGCConditions 94/1 55/2 72/1.5 30
A. bovis 16S rRNA ABKf TAGCTTGCTATGGGGACAA 547 Kang et al. (2011)AB1r TCTCCCGGACTCCAGTCTGConditions 94/0.3 59/0.3 72/0.3 25
Borrelia spp.16S rRNA
B1 CAGTGCGTCTTAAGCATGC 1,427 Part et al. (2004)B8 CCTTAAATACCTTCCTCCCConditions 94/1 58/1 72/1.5 30B3 GCAGCTAAGAATCTTCCGCAATGG 714B6 CAACCATGCAGCACCTGTATATConditions 94/0.5 59/0.75 72/0.75 25
Borrelia spp. groEL GF TACGATTTCTTATGTTGAGGG 310 Park et al. (2004)GR CATTGCTTTTCGTCTATCACCConditions 94/0.5 57/0.75 72/0.75 35
Bartonella spp. internaltranscribed spacer
QHVE1 TTCAGATGATGATCCCAAGC 735 La Scola et al. (1999)QHVE3 AACATGTCTGAATATATCTTCConditions 94/0.75 55/0.75 72/0.75 30QHVE12 GCAGCTAAGAATCTTCCGCAATGG 484 Seki et al. (2006)QHVE14 CAACCATGCAGCACCTGTATATConditions 94/0.5 59/0.75 72/0.75 30
FIG. 2. Monthly distribution of total number of captured and banded migratory birds, and number of ticks, by species,collected from the migratory birds at Hong-do Island from January, 2008, through December, 2009, Jeollanam-do Province,Republic of Korea.
TICK-BORNE PATHOGENS IN TICKS FROM MIGRATORY BIRDS 3
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DNA extraction and PCR amplification
Ticks were pooled by tick species, life stages, sex (adults),collection dates, and unique bird collection numbers for eachspecies. Ticks were homogenized mechanically using aBeadbeater TissueLyser II (QIAGEN) with 180 lL of lysisbuffer, 20 lL of proteinase K (600 AU/mL), and 5-mm stain-less steel beads at 30 frequencies/s for 5 min, followed byincubation at 56�C overnight and then centrifugation at12,000 · g for 10 min at room temperature. After centrifuga-tion, the supernatant was used for genomic DNA extractionperformed with DNeasy� Tissue Kits (QIAGEN) according tothe manufacturer’s instructions.
PCR and nested PCR were performed using specificprimers for A. phagocytophilum, A. bovis, and Bartonella andBorrelia spp. (Table 1). A. phagocytophilum genomic DNA,provided by J. Stephen Dumler ( Johns Hopkins UniversitySchool of Medicine, Baltimore, MD), was used as a positivecontrol for detecting Anaplasma species. Bartonella henselae andB. burgdorferi isolates purchased via the American Type Cul-ture Collection were used as positive controls. The first andnested PCRs were performed in a total volume of 25 lL. EachPCR mixture consisted of 10 pmol of primers, 1 U recombi-nant Taq DNA polymerase (Takara Bio, Inc.), 10 · PCR buffer(Takara Bio, Inc.), 2.5 mM deoxyribonucleotide triphosphates(dNTPs) mixture (Takara Bio, Inc.), 10- to 100-ng samples ofgenomic DNA for the first PCR, and 1 lL of the first PCRproduct for the second PCR. The amplification of PCR prod-ucts was carried out in a PTC-200 thermal cycler (MJ Re-search, Inc.) as described in Table 1.
Cloning, nucleotide sequencing, and phylogeneticanalysis
The PCR products were purified with QIAquick Gel Ex-traction kits (QIAGEN). After purification, the amplicons
were cloned with pGEM�-T Easy Vectors (Promega) followedby transformation into Escherichia coli DH5a, and then platedonto LB agar containing 50 lg/mL of ampicillin. PlasmidDNA for sequencing was purified using the Wizard� Plus SVMinipreps DNA Purification System (Promega) according tothe manufacturer’s instructions. Purified recombinant plas-mid DNA was sequenced using a T7 and SP6 promoter pri-mer set by dideoxy termination with an automatic sequencer(ABI 3730xl capillary DNA sequencer, Applied Biosystems).The obtained sequences were evaluated with Chromas soft-ware (Ver 2.33, http://www.technelysium.com.au/chromas.html/), aligned using Clustal X (Ver 2.0, http://www.clustal.org/), and examined with a similarity matrix.Relationships between individuals were assessed by theneighbor-joining (NJ) method with nucleotide distance(maximum composite likelihood) for 1,000 replications with abootstrap test. The phylogenetic tree was based on the se-quences and determined using MEGA4 program.
Nucleotide sequence accession numbers
The GenBank accession numbers of 16S ribosomal (r)RNA,groEL, and internal transcribed space (ITS) gene sequencesand specific genospecies sequences related to bacterial pathogensfor sequence comparisons are included in Figures 3–6, below.
Results
A total of 3,816 birds representing 102 species were bandedand examined for ectoparasites from January, 2008, to De-cember, 2009 (Fig. 2), and 212 ticks were removed from 65birds belonging to 21 species and 12 genera (Table 2). Themajority of ticks were collected from White’s Thrush (Zootheraaurea) (114 ticks, 53.8%), followed by the Pale Thrush (Turduspallidus) (27 ticks, 12.7%), the Yellow-throated Bunting (Em-beriza elegans) (20 ticks, 9.4%), and Naumann’s Thrush (Turdus
Table 3. Prevalence of Anaplasma phagocytophilum, A. bovis, Bartonella grahamii, and Borrelia spp.
Based on 16S rRNA Gene Targeting PCR in Ticks from Migratory Birds on Hong-do Island,
Jeollanam-do Province, Republic of Korea
No. PCR-positive pools (MLE)a
Species StageNo. pools(no. ticks)
Anaplasmaphagocytophilum
Anaplasmabovis
Borreliaspp.
Borreliaturdi
Bartonellagrahamii
Haemaphysalis flava Larvae 5 (8) 0 0 0 0 0Nymph 25 (32) 0 0 0 0 0
Haemaphysalis longicornis Larvae 2 (10) 0 0 0 0 0Nymph 2 (2) 0 0 0 0 0
Haemaphysalis ornithophila Adult 1 (1) 0 0 0 0 0
Ixodes nipponensis Larvae 2 (2) 0 0 0 0 0Nymph 10 (11) 1 (90.18) 0 1 (90.18) 1 (94.70) 0
Ixodes turdus Larvae 22 (92) 0 0 1 (10.29) 0 1 (10.29)Nymph 28 (43) 0 1 (22.85) 2 (46.25) 1 (23.40) 0Adult 11 (11) 0 0 0 1 (90.91) 0
— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —Subtotal Larvae 31 (112) 0 0 1 (8.58) 0 1 (8.58)
Nymph 65 (88) 1 (11.30) 1 (11.30) 3 (34.90) 2 (23.00) 0Adult 12 (12) 0 0 0 1 (83.33) 0
Total 108 (212) 1 (4.66) 1 (4.66) 4 (18.7) 3 (14.09) 1 (4.66)
aPCR-positive pathogens were determined by the maximum likelihood estimates (MLE), based on the number of positive individuals/pools and pool size for ticks tested.
TICK-BORNE PATHOGENS IN TICKS FROM MIGRATORY BIRDS 5
FIG. 3. (A) Phylogenetic relationships for Anaplsama phagocytophilum (boldface) detected in ticks collected from migratorybirds in the Republic of Korea and Anaplasma and Ehrlichia species based on partial nucleotide sequences of the 926-bp 16SrRNA genes. (B) Phylogenetic relationships for A. phagocytophilum (boldface) detected in ticks collected from migratory birdsand Anaplasma and Ehrlichia species based on partial protein sequences of the 409-amino-acid groEL genes. The neighbor-joining method was used for constructing a phylogenetic tree. The numbers at nodes are the proportions of 1,000 bootstrapiterations that support the topology shown.
6
naumanni) (10 ticks, 4.7%). The remaining 17 species ac-counted for 19.3% (ranging from 1 to 6 ticks, 0.5–2.8%) of thetotal number of ticks collected. A total of 5 species (larvae,nymphs, and adults) of ticks, belonging to 2 genera, wereidentified (Tables 1 and 3). The most frequently collected tickwas I. turdus Nakatsuji (146, 68.9%), followed by Haemaphy-salis flava Neumann (40, 18.9%), Ixodes nipponensis Kitaoka andSaito (13, 6.1%), Haemaphysalis longicornis Neumann (12,5.7%), and Haemaphysalis ornithophila Saito, Hoogstraal, andWassef (1, 0.5%) (Table 1).
A. phagocytophilum and A. bovis 16S rRNA genes wereidentified by species-specific nested PCR. A. phagocytophilumwas detected in I. nipponensis collected from Zoothera sibiricaand A. bovis was detected in I. turdus collected from Emberizachrysophrys (Table 3). The genome sequences were analyzedand compared with the fragments of 16S rRNA genesequences to demonstrate genetic relationships betweenAnaplasma spp. detected in ticks. The acquired A. phagocyto-philum 16S rRNA gene sequence ( JX219480) corresponded topreviously sequenced A. phagocytophilum (GU556624) fromthe ROK (Fig. 3A), whereas the acquired A. bovis 16S rRNAgene sequence ( JX219481) was 99.8% similar to A. bovis se-quence (GU556626) from the ROK (Fig. 4).
Using the groEL gene-specific primer pairs shown in Table 1for PCR, one A. phagocytophilum groEL gene nucleotide se-
quence ( JX219474) was obtained. The product size was 1,297base pairs (bp) and translated 409 amino acids of groEL. Theobtained groEL amino acid sequence was identical to A. phago-cytophilum (ADO34908) from the ROK (Fig. 3B).
Borrelia spp. (n = 4) and B. turdi (n = 3) 16S rRNA genes wereidentified by nested PCR assay. B. turdi were detected in 2 I.turdus and 1 I. nipponensis collected from Z. aurea (2) andT. pallidus (1), whereas Borrelia spp. were detected in 3 I. turdusand 1 I. nipponensis collected from Emberiza tristrami (1),T. pallidus (2), and Z. aurea (1). The genetic relationships be-tween 7 genome sequences of Borrelia spp. detected in tickswere analyzed and compared with the fragments of 16S rRNAgene sequences (Fig. 5A). The Borrelia spp. 16S rRNA genesequences ( JX219478) were identical and showed 99.9% ho-mology to Borrelia tanukii (NR025874) from Japan. The ob-tained B. turdi 16S rRNA gene sequences ( JX219479) wereidentical and showed 99.9% homology to B. turdi (NR025873and D67024) from Japan (Fig. 6).
Using the groEL specific nested PCR, 2 Borrelia spp.( JX219476) and 2 B. turdi ( JX219475) groEL gene sequenceswere detected in ticks collected from migratory birds. Theproduct size was 310 bp and contained coding sequence for103 amino acids of groEL. In spite of being characterized asdifferent species, 99.4% homology was shared between the 2Borrelia spp. and 2 B. turdi groEL genetic sequences (Fig. 5B),
FIG. 4. Phylogenetic relationships among Anaplasma bovis (boldface) detected in ticks collected from migratory birds in theRepublic of Korea and Anaplasma and Ehrlichia species based on partial nucleotide sequences of the 547-bp 16S rRNA genes.The neighbor-joining method was used to construct a phylogenetic tree. The numbers at nodes are the proportion of 1,000bootstrap iterations that support the topology shown.
TICK-BORNE PATHOGENS IN TICKS FROM MIGRATORY BIRDS 7
whereas all of the amino acid sequences were identical. TheBorrelia spp. and B. turdi groEL genes were different at 2 sites(nucleotide positions 113 and 205) of 310 nucleotide se-quences. The 2 Borrelia spp. groEL gene sequences wereidentical and showed 99.4% and 99.0% homology to B. turdi(AF517972) and B. tanukii (AF517973), respectively. Two B.turdi groEL gene sequences were identical each other andshowed 100.0% and 99.7% homology to B. turdi (AF517972)and B. tanukii (AF517973), respectively (Fig. 5B).
The B. grahamii ITS gene ( JX219477) was detected by nestedPCR assay in I. turdus collected from Z. aurea (Table 3). The
product size was 484 bp and contained noncoding sequences.To investigate the genetic relationship between the Bartonellaspecies detected in ticks, the ITS sequence was analyzed andcompared with the fragments of ITS gene sequences. The ac-quired B. grahamii ITS gene sequence was identical to B. gra-hamii (AJ269785) in UK (Fig. 6).
Discussion
The bird population of Korea is augmented by millions ofbirds that migrate or stop over from April to May and
FIG. 5. (A) Phylogenetic relationships among Borrelia tanukii and B. turdi (boldface) detected in ticks collected from mi-gratory birds in the Republic of Korea and Borrelia species based on partial nucleotide sequences of the 704-bp 16S rRNAgenes. (B) Phylogenetic relationships among Borrelia spp. (boldface) detected in ticks collected from migratory birds in theRepublic of Korea and Borrelia species based on partial nucleotide sequences of the 310-bp groEL genes. The neighbor-joiningmethod was used to construct a phylogenetic tree. The numbers at nodes are the proportion of 1,000 bootstrap iterations thatsupport the topology shown.
8 KANG ET AL.
September to November. More than 100 species of migra-tory birds are recorded annually on their spring migrationsto their summer breeding grounds in the northeastern Pa-learctic region, including Russia and eastern China, andautumn migrations to their nonbreeding grounds insoutheast Asia and Australia (Pospelova-Shtrom et al. 1965,Filippova 1984, Lee et al. 2003). These bird migrations arebehavioral responses, i.e., traversing long distances to theirbreeding and overwintering grounds that are affected bychanges in food availability, habitat, and weather. Emberiza,Zoothera, and Turdus spp. migrate annually and breed in theSiberian taiga zone during the summer and return to south-east Asia during the winter. Turdus spp. are known to haverelatively high tick infestation rates due to their habit ofspending a majority of their time on the ground in search offood among grasses and low-lying vegetation where ticks arecommonly found (Ishiguro et al. 2000, Hasle et al. 2009a, b).
I. turdus is primarily an ectoparasite of birds (Yamauchiet al. 2001). During this surveillance period, it was collectedfrom 12 of the 21 bird species identified with ticks and was themost frequently collected tick species (66.2% of all specimens).These results are similar to migratory bird surveys conductedin the eastern Shimane Prefecture, Japan, where I. turdus ac-counted for 79.0% of all ticks collected from birds (Yamauchiand Mori 2004). Haemaphysalis ornithophila is widespread insoutheast Asia and China, but is infrequently collected frombirds throughout its range. Only 1 specimen was collectedfrom a White’s Thrush (Z. aurea) during this survey, whereas 2adults were collected in previous bird banding surveys in theROK (Kim et al. 2009). Z. aurea may transport this tick, in
addition to other species, when migrating to their summerbreeding grounds in Russia (Kim et al. 2009).
A. phagocytophilum and A. bovis are obligate intracellular tick-borne bacteria in the family Anaplasmataceae with Ixodes spp.as the primary vectors. Limited studies of birds as reservoirs ofA. phagocytophilum have been conducted. A. phagocytophilumDNA was detected in I. ricinus and I. scapularis collected frommigratory birds in Canada, Norway, Poland, Russia, Spain,and Sweden (Alekseev et al. 2001, Bjoersdorff et al. 2001, Sko-tarczak et al. 2006, Ogden et al. 2008, Paulauskas et al. 2009,Hildebrandt et al. 2010, Palomar et al. 2012). These studiespresented a low prevalence of A. phagocytophilum in ticks col-lected from migratory birds, suggesting that birds were not acompetent reservoir for A. phagocytophilum. In this study, only 1A. phagocytophilum and 1 A. bovis were detected in I. nipponensisand I. turdus, respectively. The low prevalence of Anaplasmaspp. in our study was similar to previous reports (Alekseevet al. 2001, Bjoersdorff et al. 2001, Skotarczak et al. 2006, Ogdenet al. 2008, Paulauskas et al. 2009, Hildebrandt et al. 2010, Pa-lomar et al. 2012). Nevertheless, migratory birds might be animportant component in the dispersal of ticks infected withendemic Anaplasma spp. throughout their range where theyoverwinter and breed.
B. turdi has not been reported to cause disease in humansand was previously only detected in I. turdus ticks collectedfrom migratory birds in Japan (Fukunaga et al. 1996). Recentlyin Norway, Hasle et al. (2011) detected B. turdi in I. ricinuscollected from migratory birds, but failed to show the genesequences and phylogenetic analysis. In this study, Borreliaspp. sequences in I. nipponensis and I. turdus collected from
FIG. 6. Phylogenetic relationships among Bartonella grahamii (boldface) detected in ticks collected from migratory birds andBartonella species based on partial nucleotide sequences of the 484-bp internal transcribed spacer (ITS) genes. The neighbor-joining method was used to construct a phylogenetic tree. The numbers at nodes are the proportion of 1,000 bootstrapiterations that support the topology shown.
TICK-BORNE PATHOGENS IN TICKS FROM MIGRATORY BIRDS 9
migratory birds were divergent from previously reportedBorrelia spp. sequences (Fig. 5). The Hongdo-4-1 genotype( JX219475) belonged to the B. turdi clade, but was divergentfrom other reported B. turdi sequences. The Hongdo-4-4 ge-notype ( JX219476) did not separate into any of the Borreliaspp. subclades. These results and reported studies suggestthat the vector of B. turdi is I. turdus and the reservoirs areground-dwelling birds.
Fleas have been identified as the vectors for many Bartonellaspp. The rodent flea (Ctenocephalides nobilis nobilis) has beenidentified as the primary vector of B. grahamii (Bown et al.2004). More recently a number of tick species have been shownto be positive for Bartonella spp. based primarily on PCRtechniques or infrequently by culture (Billeter et al. 2008). In theROK, Bartonella DNA has been detected in H. flava, H. long-icorinis, I. nipponensis, and I. turdus ticks collected from rodentsor by tick drag (Kim et al. 2005). In this study, the B. grahamiiITS gene sequence ( JX219477) was detected in I. turdus tickscollected from the Pale Thrush (T. pallidus). However, therewere insufficient ITS gene sequences available for a compre-hensive comparison with sequences reported in GenBank.Additional phylogenetic analysis for gltA, groEL, and rpoBgenes targeting nested PCR was conducted, but unfortunatelya positive band was not obtained. Nevertheless, this result in-dicates that I. turdus and migratory birds may be potentialvectors and reservoirs of Bartonella spp. in Asia.
Hard ticks are the primary vectors of a variety of bacterialpathogens, including Anaplasma, Borrelia, and Ehrlichia spp.(Parola et al. 2001). Although Bartonella spp. are transmittedby other ectoparasitic arthropods, the only evidence to sup-port the possibility of tick-borne transmission is indirect(Angelakis et al. 2010). Recently, because of the rapidly ex-panding number of reservoir-adapted pathogens that havebeen discovered, efforts to clarify the vector competence,potential for transmission, and disease health risks are rele-vant to public and veterinary health. Herein, we present thevarious tick-borne pathogens, i.e., Anaplasma, Bartonella, andBorrelia spp. that were detected in Ixodes spp. from migratorybirds. These results do not conclusively identify the role ofmigratory birds as reservoirs or introduction of exotic ticks innonendemic areas, but they provide an insight into the po-tential role of migratory birds in the dispersal and infectiouscycles of tick-borne pathogens in Asia.
Acknowledgments
This research was supported by a National ResearchFoundation of Korea Grant funded by the Korean Govern-ment (2011-0015349), and funding for portions of this workwas provided by the Armed Forces Health SurveillanceCenter, Global Emerging Infections Surveillance and Re-sponse System, Silver Spring, MD, and the National Centerfor Military Intelligence, Ft. Detrick, MD. We thank themembers of Migratory Bird Center, National Park ResearchInstitute, for bird tick collections during their bird-bandingsurveys. We especially thank Dr. Joel Gaydos, Global Emer-ging Infections Surveillance and Response System, SilverSpring, MD, USA, for his support and constructive criticism.
Author Disclosure Statement
The opinions expressed in this article are those of the au-thors and do not reflect official policy or positions of the US
Department of the Army, the US Department of Defense, orthe US Government.
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Address correspondence to:Joon-Seok Chae
Laboratory of Veterinary Internal MedicineCollege of Veterinary Medicine
Seoul National UniversitySeoul, 151-742
Korea
E-mail: [email protected]
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