Identification of Ehrlichia chaffeensis , Anaplasma phagocytophilum , and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus Ticks from Korea

VECTOR-BORNE AND ZOONOTIC DISEASESVolume 3 Number 1 2003copy Mary Ann Liebert Inc

Identification of Ehrlichia chaffeensis Anaplasmaphagocytophilum and A bovis in Haemaphysalis longicornis

and Ixodes persulcatus Ticks from Korea

CHUL-MIN KIM1 MIN-SEOK KIM1 MI-SUN PARK2

JIN-HO PARK1 and JOON-SEOK CHAE1

ABSTRACT

A total of 1467 tick (1463 of Haemaphysalis longicornis three of Ixodes persulcatus and one of I turdus) collectedfrom nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia andAnaplasma species One set of primers and a probe were designed for detection of all of the Ehrlichia andAnaplasma species Template DNAs (total 803) were prepared either from pools of larvae nymphs adult malesand females or from the salivary gland and midgut of adult ticks Only DNAs positive in TaqMan PCR were ex-amined for A phagocytophilum with nested PCR and for E chaffeensis with PCR Four A phagocytophilum 16SrRNA gene PCR products were sequenced for comparison with sequences previously reported Amplification ofa 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (453 of the to-tal) Of these 364 positive ticks species-specific PCRs confirmed that 35 H longicornis and one I persulcatuswerepositive for A phagocytophilum and one I persulcatus was positive in E chaffeensis Except for one (AB-GGHLGenBank accession number [GAN] AF470698) three of the four 16S rRNA gene fragment sequences of the Aphagocytophilum-positive samples were similar or identical to the sequences of variants of A phagocytophilumdeposited in GenBank The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma(Ehrlichia) bovis 16S rRNA (GAN U03775) The identities of the Anaplasmataceae genus and species DNA in the327 ticks that could not be confirmed infected with either E chaffeensis A phagocytophilum or A bovis are notknown This study is the first to demonstrate the presence of E chaffeensis A phagocytophilum and A bovisin Korean ticks Key words AnaplasmataceaemdashErlichia chaffeensismdashAnaplasma phagocytophilummdashAnaplasmabovismdashTicksmdashKorea Vector-Borne Zoonotic Dis 3 17ndash26 2003

17

INTRODUCTION

MEMBERS OF THE FAMILY Anaplasmataceae areobligatory intracellular gram negative

bacteria that parasitize monocytesmacro-phages granulocytes erythrocytes endothelialcells or platelets and cause noncontagiousfebrile systemic illnesses variably called ehrli-chiosis in humans and in some varieties of do-mestic and wild animals or anaplasmosis in ru-

minants (Murphy et al 1998 Pusterla et al1999a Foley et al 1999 Chae et al 2000 Shi-bata et al 2000 Walls 2000) The genus Ehrlichiawas divided into three distinct clusters basedon the nucleotide sequence homology of the16S rRNA genes (Anderson et al 1993 Baum-garten et al 1999 Dawson et al 1996 Rikihisa1991) However members of the Ehrlichiaphagocytophilum group are genetically and bio-logically indistinguishable and most authori-

1Bio-safety Research Institute College of Veterinary Medicine Chonbuk National University Jeonju Jeonbuk Korea

2Mol-BioNet Research Institute Scientec Lab Center Co Ltd Daejon Korea

ties now accept that these three bacteria com-prise a single species (Dumler et al 2001 Sum-ner et al 1997) Thus the E phagocytophilagroup as well as Ehrlichia bovis and Ehlrichiaplatys are now classified within the genusAnaplasma because of their close relationship toAnaplasma marginale by phylogenetic studiesusing the 16S rRNA gene and groESL (Dumleret al 2001 Fuente et al 2001)Anaplasmataceae are transmitted to humans

and animals by specific species of ticks fromwild-animal reservoirs (Barlough et al 1997abBreitschwerdt et al 1998 Cao et al 2000 Lizet al 2000 Pusterla et al 1999b Pusterla et al1999c) E chaffeensis has been found in Ambly-omma americanum (Lone star tick) A testudi-narium Haemaphysalis yeni and H flava (Cao etal 2000) A phagocytophilum has been identi-fied not only in Ixodes scapularis and Ixodes paci-ficus ticks but also in Ixodes ricinus (sheep tick)(Barlough et al 1997a Pusterla et al 1999aSchouls et al 1999) The diagnosis of diseasescaused by these bacteria is based on clinicalsigns laboratory findings tick infestation sea-son and geographical location (Barlough et al1996 Barlough et al 1997a Rikihisa 1991Walker et al 1996) Laboratory detection meth-ods include PCR amplification of ehrlichialDNA in blood of suspected hosts or vectors(Breitschwerdt et al 1998 Georges et al 2001Massung et al 1998) PCR makes it possible toidentify the presence of bacterial DNA in cul-ture-negative samples from patients with sus-pected infection (Pusterla et al 2000) Com-petitive PCR is a standard method for thequantification of DNA and has been used suc-cessfully in a number of studies However thistechnique is labor intensive and requires re-sults from multiple reactions to be analyzed foreach sample The causative agents of ehrli-chiosis in humans and animals were detectedfrom many kinds of ticks around the worldbut they have not yet been identified in KoreaHeo et al (2002) reported that detection of Echaffeensis and A phagocytophilum antibody inhuman sera by IFA test and Western im-munoblot blot analysis Therefore we col-lected 799 longicornis three I persulcatus andone I turdus ticks from nine provinces of Ko-rea to identify Ehrlichia and Anaplasma speciesby TaqMan PCR followed by in-depth exami-

nation of TaqMan PCR-positive DNA samplesfor A phagocytophilum and E chaffeensis by PCRmethods and sequencing

MATERIALS AND METHODS

Tick collection

Ticks were collected by dragging a flannelcloth over vegetation or by removing attachedticks on host animals such as cattle horsesgoats dogs cats hedgehogs and wild miceTicks were identified to the species level andto developmental stage A total of 1467 ticksmdash1463 longicornis three I Persulcatus and one Iturdusmdashwere collected from nine provinces(Gangwon Gyeonggi Chungbuk ChungnamGyeongbuk Gyeongnam Jeonbuk Jeonnamand Jeju) of Korea A total of 102 adult femaleticks collected from Jeju province were usedto obtain salivary gland and midgut They werestored in 15 mL Eppendorf tubes at 220degC in-dividually until DNA extraction was per-formed

DNA extraction

Each individual adult and nymph tick (total664) or 139 pools of larvae (5ndash10 ticks per pool)in Eppendorf tubes was mechanically homog-enized using sterile scissors 102 additionaladult female ticks were used to obtain one ofthe two salivary glands and the midgut for PCRamplification DNA was extracted with the QIAamp tissue kit (Qiagen Valencia CA) ac-cording to the manufacturerrsquos instructions

TaqMan real-time and conventional polymerasechain reaction

The approach applied for characterization ofEhrlichia and Anaplasma species was to screenall ticks and selected tick tissues with the useof a highly sensitive real time polymerase chainreaction (PCR) based upon the TaqMan systemfor the presence of 16S rRNA genes of Anaplas-mataceae Tick samples that were positive in thissensitive but broadly reactive TaqMan assaywere subjected to analysis by PCR to identifythe presence of either E chaffeensis (nadA PCR)or A phagocytohilum (nest PCR) Some ampli-

KIM ET AL18

19DETECTION OF EHRLICHIA AND ANAPLASMA IN KOREAN TICKS

TABLE1

RESU

LTS

OFTAQM

ANREAL-T

IMEPCR F

OR

THED

ETECTIO

NOFEHRLICHIA

ANDANAPLASMA

SPP I

NW

HOLETIC

KS

ANDTHEIR

ORGANS

(SALIV

ARYG

LAND

ANDM

IDGUT) C

OLLECTED

FROM

KOREA

No of tick DNAs with positive reactionstotal ()

Adult females

Larval

Adult

Whole

Salivary

Unclassified

Provinces

poolsa

Nymphs

males

ticks

gland

Midgut

ticks

Total

Gan

gwon

00

01

10 (10

)0

00

110

(10

)Gye

ongg

i75

122

(61

5)

48c 111 (432)

66b

93 (71

)53

b 85

(62

4)

00

024

241

1 (589)

Chu

ngbu

k0

621 (28

6)

24 (50)

215

(13

3)

00

010

40

(25

)Chu

ngna

m2

10 (20

)0

00

00

02

10 (20

)Gye

ongb

uk

00

07

20 (35

)0

00

720

(35

)Gye

ongn

am3

3 (100

)15

20 (75

)2

2 (100

)0

00

020

25

(80

)Jeon

buk

04 (0)

19 (111)

07

20 (35

)0

08

63 (12

7)

1696

(16

7)

Jeon

nam

08

10 (80

)0

310

(30

)0

00

12 (0)

1132

(34

4)

Jeju

00

01120

(55

)18

75

(24

)19

27

(70

4)

737

(18

9)

5515

9 (346)

Total

801

39 (57

6)

78171 (456)

7099

(70

7)

8418

0 (467)

1875

(24

)19

27

(70

4)

1511

2 (134)

364

803 (453)

a 5ndash10 tick

s per poo

lbInclud

ing on

e Ixodes persulcatus

c Inc

luding on

e Ixodes turdus

cons that were not amplified with eitherspecies-specific method were analyzed by di-rect sequencing

The TaqMan primers ESP-F (59-agtccacgctg-taaacgatgag-39) ESP-R (59-ttcctttgagttttagtctt-gcgac-39) and probe ESP-P (59-69-FAM-acgcgt-taagcactccgcctgg-TAMRA-39) which specifi-cally targets the 16S rRNA gene of Ehrlichia andAnaplasma species were designed using thePrimer Express software program (PE AppliedBiosystems) according to the manufacturerrsquosinstructions The TaqMan PCR mixture (20 mLtotal volume) consisted of 450 nM of eacholigonucleotide primer 250 nM fluorescent la-beled probe and TaqMan Universal PCR Mas-ter Mix (PE Applied Biosystems USA) per re-action All of the PCR reactions were mixed in96-well optical plates and cycled in SDS 7700under the following conditions 50degC for 2 min(uracil N-deglycosylase digest) 95degC for 10 min(AmpliTaq Gold pre-activation) and 45 cyclesof 95degC for 20 sec and 55degC for 60 sec The SDS7700 cycler provided cycle-by-cycle measure-ment of the fluorescence emission from eachPCR reaction The fluorescence data wasanalysed using Sequence Detector program(Ver 17 ABI)

PCR for E chaffeensis was performed usingprimers ECH-F (59-ATATGATGGTTAGA-CATGATAAAGCATATG-39) and ECH-R (59-

TTTCATTGGATCGACAACTCAGTTA-39)(101 bp) derived from the nadA gene of E chaf-feensis (Yu et al 1997) For amplification 50-100ng of each template sample was amplified in25mL reaction mixture The thermal cyclingprotocol was as follows pre-annealing denat-uration for 5 min at 94degC followed by 45 cy-cles of 94degC for 1 min 55degC for 30 sec and 72degCfor 45 sec followed by final extension at 72degCfor 7 min The 16S rRNA gene fragment of Aphagocytophilum was amplified by nested PCRaccording to the procedure of Barlough et al(1996) For comparison of nucleotide sequencesamong Ehrlichia or Anaplasma 16S rRNA genefragments (116 bp) E chaffeensis and A phago-cytophilum DNAs were amplified by PCR withthe ESP primers

Specificity test for TaqMan real-time polymerasechain reaction

Positive and negative DNAs from organismsof related tick-borne diseases were tested to ex-amine the specificity of the probe and primersdesigned for Ehrlichia and Anaplasma speciesThe samples tested and their sources were asfollows A phagocytophilum (HGE agent twoCalifornia isolates and one isolate provided byJ Stephen Dumler Johns Hopkins UniversitySchool of Medicine Baltimore MD) A phago-

KIM ET AL20

FIG 1 PCR amplification of a 101-bp E chaffeensis nadA gene fragment from Korean ticks M 100-bp size marker(iNtRON Biotechnology Korea) N negative (water) control P positive control (Johns Hopkins University School ofMedicine) lane 1 2 4 5 E chaffeensisndashnegative samples 3 E chaffeensisndashpositive sample (Ixodes persulcatus fromGyeonggi)

FIG 2 Nested PCR amplicons of a 926-bp A phagocytophilum 16S rRNA gene fragment from Korean ticks M 100-bp size marker (iNtRON Biotechnology Korea) N negative (water) control P positive control (CA isolate) lane 12 6 7ndash12 A phagocytophilumndashnegative samples lane 3ndash5 13ndash14 A phagocytophilumndashpositive samples

cytophilum (E equi Nantucket Island isolateMRK strain and CA isolate) E chaffeensisArkansas strain (J Dawson CDC AtlantaGA) Neorickettsia (Ehrlichia) risticii (CaliforniaUSA isolate) A marginale (Dr Hee-jong SongKorean isolate) Babesia bovis (Dr Gale WagnerTexas AampM University) B gibsoni (JeonbukKorean isolate) B odocoilei (Texas USA iso-late) B caballi (Texas USA isolate) Theileriaequi (Texas USA isolate) T sergenti (Jeju Ko-rean isolate) Theileria sp MSD (South Africanisolate) and Borrelia burgdorferi (California USAisolate) Tick bovine equine and human ge-nomic DNAs were used in PCR for negativecontrol template

Comparison of sensitivity between conventionaland TaqMan real-time polymerase chain reaction

A phagocytophilumndash and E chaffeensisndashposi-tive DNAs were used for comparing the sensi-tivity of conventional and TaqMan PCR in therange from 102 ngmL to 1027 ngmL (10-foldserial dilution) Conventional PCR was per-formed with 10-fold serial dilutions of the DNAas the template in the following four reactionsfor A phagocytophilum 16S rRNA gene frag-ment (926 bp) according to Barlough et al(1996) for E chaffeensis nadA gene fragment(101 bp) with E chaffeensis specific primers(ECH primer) for A phagocytophilum 16SrRNA gene fragment (116 bp) with Ehrlichiaand Anaplasma genus specific primers (ESP

primer) and for E chaffeensis 16S rRNA genefragment (116 bp) with ESP primers

Nucleotide sequencing and analysis

PCR products were purified by QIAquickPCR Purification Kit (Qiagen Valencia CA) be-fore sequencing using PE Biosystems Prism 377automatic DNA Sequencer Nucleotide se-quence homology searches were made throughthe National Center for Biotechnology Infor-mation (NCBI National Institute of Health)BLAST network service Nucleotide sequencesamong Ehrlichia and Anaplasma species werecompared by MultAlign program (wwwtoulouseinrafrlgcmultalinmultalinhtml)The percent of identity to the sequences ofEhrlichia and Anaplasma species was performedby using sequence data program ALIGNQuery (Genestream France httpxylianighcnrsfrbinalign-guesscgi)

RESULTS

Of the positive and negative DNAs fromagents of related tick-borne diseases that wereexamined for analysis of specificity onlyEhrlichia and Anaplasma species were amplifiedby TaqMan PCR The positive DNAs were asfollows all three isolates of A phagocytophilumHGE agent examined all three isolates of Aphagocytophilum (E equi) the E chaffeensis


TABLE 2 RESULTS OF NESTED PCR FOR THE DETECTION OF A PHAGOCYTOPHILUM IN WHOLE TICKS AND THEIR ORGANS

(SALIVARY GLAND AND MIDGUT) COLLECTED FROM KOREA

No of tick DNAs with positive reactionstotal ( positive)

Adult females

Larval Adult Whole Salivary UnclassifiedProvinces pools Nymphs males ticks gland Midgut ticks Total

Gangwon 0 0 0 01 (0) 0 0 0 01 (0)Gyeonggi 075 (0) 048 (0) 7a66 (106) 053 (0) 0 0 0 07242 (29)Chungbuk 0 36 (50) 02 (0) 02 (0) 0 0 0 310 (30)Chungnam 02 (0) 0 0 0 0 0 0 02 (0)Gyongbuk 0 0 0 27 (286) 0 0 0 27 (286)Gyongnam 03 (0) 315 (20) 02 (0) 0 0 0 0 0320 (15)Jeonbuk 0 01 (0) 0 27 (286) 0 0 08 (0) 216 (125)Jeonnam 0 08 (0) 0 0 0 0 03 (0) 011 (0)Jeju 0 0 0 211 (182) 018 (0) 1519 (789) 27 (286) 1955 (345)Total 080 (0) 678 (77) 770 (10) 684 (71) 018 (0) 1519 (789) 215 (133) 36364 (99)

aIncluding one Ixodes persulcatus

Arkansas strain the Neorickettsia (Ehrlichia) ris-ticii California isolate) and the A marginale Ko-rean isolate TaqMan PCR detected 1022 ngmLof A phagocytophilum and E chaffeensis DNA af-ter 45 cycles The samples containing less than1022 ngmL and the no-template control gavenegative results (data not shown) PCR for Echaffeensis with ECH primers and A phagocy-tophilum and E chaffeensis with ESP primers detected 102 ngmL to 100 ngmL Nested PCRdetected 102 ngmL to 1022 ngmL of A phago-cytophilum DNA

Infection in whole crushed ticks or ticksali-vary gland and midgut varied from 10 to80 with a mean of 453 Eighteen (24) and19 (704) ticks examined were also positive insalivary gland and midgut respectively (Table1) Anaplasmataceae-positive DNAs were thenexamined using nested PCR for A phagocy-tophilum and nadA PCR for E chaffeensis Of the364 ticks examined by species-specific PCR the

nadA gene fragment of E chaffeensis was iden-tified by PCR in only one I persulcatus tick (Fig1) Similarly a 16S rRNA gene fragment of Aphagocytophilum was amplified by nested PCR(Fig 2) in 36 ticks collected from GyeonggiChungbuk Gyongbuk Gyongnam Jeonbukand Jeju with a positive rate of 99 (36364)for whole ticks and 789 (1519) when midgutDNA was used as the template (Table 2) The16S rRNA gene fragment sequences (PCR withESP primer 116 bp) of three randomly selectedTaqMan PCR-positive ticks that were negativein PCR for A phagocytophilum and E chaffeen-sis were identical to each other Although thesesequences were most closely related to thegenus Ehrlichia they were different from se-quences of all Ehrlichia and Anaplasma speciesreported previously (Fig 3)

Two of the four 16S rRNA gene fragment sequences of the A phagocytophilumndashpositivesamples were clearly similar to the sequences

KIM ET AL22

FIG 3 Comparison of nucleotide sequences of Ehrlichia or Anaplasma 16S rRNA gene fragments (ESP-CNHL ESP-CBHL ESP-GGHL) amplified by PCR with Ehrlichia and Anaplasma genus specific primers (ESP primer) E chaffeen-sis U60476 E muris U15527 E canis U26740 E ewingii M73227 A phagocytophila M73224 A playts AF286699 A cen-trale AF283007 A bovis U03775

of the other variants of A phagocytophilumCompared to Anaplasma sp LGE isolate (GANAF241532) the sequences of AP-JJHL (GANAF470700) and AP-CBHL (GAN AF470699)were 998 and 989 similar respectivelyOne of the four samples sequenced (AP-GGIPGAN AF470701) was identical to Anaplasmasp LGE isolate (GAN AF241532) However AB-GGHL (GAN AF470698) from Gyeonggiprovince was similar (988) to A bovis (GANAF294789) (Table 3) The samples sequencedand their sources are shown in Table 4 Figure4 shows the provinces where Ehrlichia orAnaplasma species Anaplasma phagocytophilumEhrlichia chaffeensis and Anaplasma bovis weredetected in Korean ticks

DISCUSSION

In 1994 and 1987 human granulocytic andmonocytic ehrlichiosis were initially describedrespectively (Chen et al 1994 Maeda et al1987) Since the first reports these diseases orserologic evidence of infection has been re-ported in more than 30 states of the UnitedStates (Walker et al 1996) as well as in Europe(Liz et al 2000 Morais et al 1991 Ravyn et al1999) Africa (Uhaa et al 1992) and Asia (Caoet al 2000 Cao et al 2000 Heppner et al 1997

Heo et al 2002 Inokuma et al 2001 Shibata et al 2000) In 1998 the DNA of the genusEhrlichia was detected in ticks collected fromGangwon Korea by PCR with genus specificprimers which amplified the 16S rRNA se-quence for all the Ehrlichia species Howeverthe DNAs considered Ehrlichia species positivedid not give any amplified product with Aphagocytophilumndashspecific primers (Kim et al1998)

This study demonstrates the presence of Aphagocytophilum and E chaffeensis in Haema-physalis longicornis Ixodes persulcatus and I tur-dus ticks from Korea One of the two I persul-catus collected was infected with E chaffeensisand the other with A phagocytophilum I scapu-laris and I pacificus in the United States and Iricinus ticks in many European countries havebeen identified as potential vectors of A phago-cytophilum (Barlough et al 1996 Barlough et al1997a Liz et al 2000 Richter et al 1996 Baum-garten et al 1999 Pusterla 1999a) However Hlongicornis has not been identified as a vectorof ehrlichiae although these ticks are exten-sively distributed in Korea The presence of Aphagocytophilum and E chaffeensis suggests thatehrlichiosis in both humans and animals mayoccur in Korea an hypothesis that needs to beconfirmed

In the study presented here a real-time PCR


TABLE 3 HOMOLOGY COMPARISON OF THE A PHAGOCYTOPHILUM 16S RRNA GENE FRAGMENT (926 BP) SEQUENCES

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85

Percent identity between sequences of 16S rRNA gene fragment is shown as the upper matrix The lower matrixshows the number of necleotide differences 1 AP-JJHL (GAN AF470700) 2 AB-GGHL (GAN AF470698) 3 AP-CBHL(GAN AF470699) 4 AP-GGIP (GAN AF470701) 5 A phagocytophilum LGE agent (AF241532) 6 A phagocytophilumJena 8 (AJ313513) 7 A phagocytophilum (M73244) 8 Anaplasma platys (AF286699) 9 Anaplasma marginale (AF311303)10 Ehrlichia chaffeensis (U60476) 11 Ehrlichia muris (U15527) 12 Ehrlichia canis (U26740) 13 Ehrlichia (Cowdria) rumi-nantium (U03776) 14 Anaplasma (Ehrlichia) bovis (U03775)

was used for detection and identification of allthe Ehrlichia and Anaplasma species in ticksTaqMan PCR was 100-fold more sensitive thanconventional PCR However the sensitivitiesof nested PCR and TaqMan PCR were identi-cal Analysis showed a high rate of infection inthe ticks with at least one Ehrlichia or Anaplasmaspecies (453) A phagocytophilum was identi-fied in 35 H longicornis and one I persulcatusE chaffeensis was found in one I persulcatuswhich is a known vector of A phagocytophilumin China (Cao et al 2000) The high infectionrate in A phagocytophilum nested PCR of

midgut (789) may be partly due to the factthat the ticks originated from an individualhost or from the same place where the infec-tion rate is high Three of four PCR productsanalysed revealed that the 16S rRNA gene se-quences of A phagocytophilum showed slightvariation or were identical to other Anaplas-mataceae In particular one sample (GANAF470701) analyzed was highly similar to anA bovis sequence in the GenBank The differ-ence was in the primer site and we did not ex-amine whether primers hybridized and an-nealed to A bovis in spite of their different

KIM ET AL24

TABLE 4 SAMPLE NAMES PROVINCES TICK SPECIES AND SITE OF TICK COLLECTION FOR IDENTIFICATION OF EHRLICHIAOR ANAPLASMA SPECIES

Ehrlichia or Host animalNames Anaplasma strain Provinces Tick species or grass

EC-GGIP E chaffeensis Gyeonggi Ixodes persulcatus GrassAP-JJHL A phagocytophilum Jeju Haemaphysalis longicornis HorseAP-GGIP A phagocytophilum Gyeonggi Ixodes persulcatus GrassAP-CBHL A phagocytophilum Chungbuk Haemaphysalis longicornis CattleAB-GGHL A bovis Gyeonggi Haemaphysalis longicornis HedgehogESP-CNHL Ehrlichia sp Chungnam Haemaphysalis longicornis CattleESP-CBHL Ehrlichia sp Chungbuk Haemaphysalis longicornis GrassESP-GGHL Ehrlichia sp Gyeonggi Haemaphysalis longicornis Cat

FIG 4 Map of south Korea showing provinces where Ehrlichia or Anaplasma species Anaplasma phagocytophilumEhrlichia chaffeensis and Anaplasma boviswere detected from Korean ticks by PCR (Ehrlichia or Anaplasma species Taq-Man PCR Anaplasma phagocytophilum and Anaplasma bovis nested PCR Ehrlichia chaffeensis conventional PCR)

Ehrlichia or Anaplasma spp

Ehrlichia chaffeensis

Anaplasma phagocytophilum(HGE)

Anaplasma bovis

sequences It is possible that a unique A bovisndashlike organism exists in Korea

Of great interest is the overall high frequencywith which Anaplasmataceae were detected inKorean ticks even in the absence of a specificidentification to the genus or species level Al-though the few sequences obtained from lim-ited 16S rRNA gene analysis indicate a closerrelationship of some of the unidentifiedAnaplasmataceae to the Ehrlichia genus the in-ability of amplification by E chaffeensis nadAPCR indicates the uniqueness of this organismFuture studies should focus upon establishingthe identity of this bacterium and determiningwhether this is a potential anthropozoonoticagent

The nadA gene is very similar among E chaf-feensis E muris and E canis We had designedPCR primers from E chaffeensis nadA gene Theamplified PCR products were tried for se-quencing but unfortunately we did not get anysequencing results Therefore we tried to se-quence for 16S rRNA gene fragment (396 bp)and the sequences were identical to E chaffeen-sis Arkansas and Sapulpa strains (data notshown)

These findings suggest that a diversity ofAnaplasmataceae may be present in Korea andthat evidence of human and animal infectionsshould be sought TaqMan PCR can be used for the rapid and accurate identification ofehrlichiae in ticks On the basis of this studythe possible occurrence of ehrlichiosis shouldbe considered in the differential diagnosis offebrile patients with a history of tick bites inKorea Moreover it is necessary to identifyother Ehrlichia and Anaplasma species that mayoccur apart from A phagocytophilum and E chaf-feensis in Korea

ACKNOWLEDGMENTS

We thank Seok-Gon Park DVM in the Na-tional Jeju Agricultural Experiment StationLivestock Division Dr Hee-Jong Song DVMin Chonbuk National University Dong-GyunNa DVM Il-Geo Jung DVM and Dr In-Young Lee for assistance in tick collection andDrs JS Dumler and SD Waghela for review-ing an early draft of the manuscript This studywas supported by a grant of the Korea Health

21 RampD Project Ministry of Health amp WelfareRepublic of Korea (HMP-00-B-20200-0003)

REFERENCES

Anderson BE Sims KG Olson JG et al Amblyommaamericanum a potential vector of human ehrlichiosisAm J Trop Med Hyg 1993 49239ndash244

Barlough JE Madigan JE Derock E et al Nested poly-merase chain reaction for detection of Ehrlichia equi ge-nomic DNA in horses and ticks (Ixodes pacificus) VetParasitol 1996 63319ndash329

Barlough JE Madigan VL Kramer JR et al Ehrlichiaphagocytophila genogroup rickettsiae in Ixodide ticksfrom California collected in 1995 and 1996 J Clin Mi-crobiol 1997a 352018ndash2021

Barlough JE Madigan JE Turoff DR et al An Ehrlichiastrain from a liama (Lama glama) and liama-associatedticks (Ixodes pacificus) J Clin Microbiol 1997b 351005ndash1007

Baumgarten BU Rollinghoff M Bogdan C Prevalenceof Borrelia burgdorferi and granulocytic and monocyticEhrlichiae in Ixodes ricinus ticks from southern Ger-many J Clin Microbiol 1999 373448ndash3451

Breitschwerdt EB Hegarty BC Hancock SI Sequentialevaluation of dogs naturally infected with Ehrlichia ca-nis Ehrlichia chaffeensis Ehrlichia equi Ehrlichia ewingii orBartonella vinsonii J Clin Microbiol 1988 362645ndash2651

Cao WC Gao YM Zhang PH et al Identification ofEhrlichia chaffeensis by nested PCR in ticks from south-ern China J Clin Microbiol 2000 382778ndash2780

Cao WC Zhao QM Zhang PH et al GranulocyticEhrlichiae in Ixodes persulcatus ticks from an area inChina where lyme disease is endemic J Clin Microbiol2000 384208ndash4210

Chae JS Foley JE Dumler JS et al Comparison of thenucleotide sequences of 16S rRNA 444 Ep-ank andgroESL heat shock operon genes in naturally occurringEhrlichia equi and human granulocytic ehrlichiosisagent isolates from Northern California J Clin Micro-biol 2000 381364ndash1369

Chen SM Dumler JS Bakken et al Identification of agranulocytotropic Ehrlichia species as the etiologic agentof human disease J Clin Microbiol 1994 32589ndash595

Dawson JE Warner CK Baker V et al Ehrlichia-like 16SrRNA sequence from wild white-tailed deer (Odocoileusvirginianus) J Parasitol 1996 8252ndash58

Dumler JS Barbet AF Bekker CP et al Reorganizationof genera in the families Rickettsiaceae and Anaplasmat-aceae in the order Rickettsiales unification of somespecies of Ehrlichia with Anaplasma Cowdria withEhrlichia and Ehrlichiawith Neorickettsia descriptions ofsix new species combinations and designation ofEhrlichia equi and ldquoHGE agentrdquo as subjective synonymsof Ehrlichia phagocytophila Int J Syst Evol Microbiol2001 512145ndash2165

Foley JE Crawford-Miksza L Dumler JS et al Humangranulocytic ehrlichiosis in Northern California twocase descriptions with genetic analysis of the EhrlichiaeClin Infect Dis 1999 29388ndash392

DETECTION OF EHRLICHIA AND ANAPLASMA IN KOREAN TICKS 25

Fuente JDL Van Den Bussche RA Kocan KM Molecu-lar phylogeny and biogeography of north american isolates of Anaplasma marginale Vet Parasitol 2001 9765ndash76

Georges K Loria GR Riili S et al Detection ofhaemoparasites in cattle by reverse line blot hybridiza-tion with a note on the distribution of ticks in SicilyVet Parasitol 2001 99273ndash286

Heo EJ Park JH Koo JR et al Serologic and moleculardetection of Ehrlichia chaffeensis and Anaplasma phago-cytophila (human granulocytic ehrlichiosis agent) in Ko-rean patients J Clin Microbiol 2002 403082ndash3085

Heppner DG Wongsrichanalai C Walsh DS et al Hu-man ehrlichiosis in Thailand Lancet 1997 350785ndash786

Inokuma H Parola P Raoult D et al Molecular surveyof Ehrlichia infection in ticks from animals in Yam-aguchi Prefecture Japan Vet Parasitol 2001 99335ndash339

Kim JB Song HW Park SU et al Detection of Borreliaburgdorferi and ehrlichiosis agent in ticks collected inKorea using polymerase chain reaction Korean J Bio-med Lab Sci 1998 4113ndash120

Liz JS Anderes L Sumner JW et al PCR detection ofgranulocytic Ehrlichiae in Ixodes ricinus ticks and wildsmall mammals in western Switzerland J Clin Micro-biol 2000 381002ndash1007

Lockhart JM Davidson WR Stallknecht DE et al Natural history of Ehrlichia chaffeensis (RickettsialesEhrlichiae) in the predominent physiographic provinceof Georgia J Parasitol 1997 83887ndash894

Maeda D Markowitz N Hawley R et al Human in-fection with Ehrlichia canis a leukocytic rickettsia NEngl J Med 1987 316853ndash856

Magnarelli LA Stafford KC Mather TN et al Hemo-cytic rickettsia-like organisms in ticks serologic reac-tivity with antisera to Ehrlichiae and detection of DNAof agent of human granulocytic ehrlichiosis by PCR JClin Microbiol 1995 332710ndash2714

Massung RF Slater K Owens JH et al Nested PCR as-say for detection of granulocytic Ehrlichiae J Clin Mi-crobiol 1998 361090ndash1095

Morais JD Dawson J Greene C et al First europeancase of ehrlichiosis Lancet 1991 388633ndash634

Murphy GL Ewing SA Whitworth LC et al A molec-ular and serologic survey of Ehrlichia canis E chaffeen-sis and E ewingii in dogs and ticks from OklahomaVet Parasitol 1998 79325ndash339

Oleksiewicz MB Donaldson AI Alexandersen S De-velopment of a novel real-time RT-PCR assay for quan-titation of foot-and-mouth disease virus in diverseporcine tissues J Virol Methods 2001 9223ndash35

Pusterla N Huder JB Leutenegger CM et al Quantita-tive real-time PCR for detection of members of theEhrlichia phagocytophila genogroup in host animals andIxodes ricinus ticks J Clin Microbiol 1999a 371329ndash1331

Pusterla N Leutenegger CM Chae JS et al Quantita-tive evaluation of the Ehrlichial burden in horses afterexperimental transmission of human granulocyticEhrlichia agent by intravenous inoculation with infected

leukocytes and by infected ticks J Clin Microbiol 1999b374042ndash4044

Pusterla N Leutenegger CM Huder JB et al Evidenceof the human granulocytic ehrlichiosis agent in Ixodesricinus ticks in Switzerland J Clin Microbiol 1999c371332ndash1334

Pusterla N Madigan JE Asanovich KM et al Experi-mental inoculation with human granulocytic Ehrlichiaagent derived from high- and low-passage cell culturein horses J Clin Microbiol 2000 381276ndash1278

Ravyn MD Korenberg EI Oeding JA et al MonocyticEhrlichia in Ixodes persulcatus ticks from Perm RussiaLancet 1999 353722ndash723

Richter PJ Kimsey RB Madigan JE et al Ixodes pacifi-cus (Acari Ixodidae) as a vector of Ehrlichia equi (Rick-ettsiales Ehrlichieae) J Med Entomol 1996 331ndash5

Rikihisa Y The tribe Ehrlichiae and ehrlichial diseases JClin Microbiol 1991 4286ndash308

Rikihisa Y Clinical and biological aspects of infectionscaused by Ehrlichia chaffeensis Microb Infect 1999 31ndash10

Roland WE Everett ED Cyr TL et al Ehrlichia chaffeensisin Missouri ticks Am J Trop Med Hyg 1998 59641ndash643

Schouls LM Van de pol I Rijpkema SGT et al Detec-tion and identification of Ehrlichia Borrelia burgdorferisensu lato and Bartonella species in Dutch Ixodes rici-nus ticks J Clin Microbiol 1999 372215ndash2222

Shibata SI Kawahara M Rikihisa Y et al New Ehrlichiaspecies closely related to Ehrlichia chaffeensis isolatedfrom Ixodes ovatus ticks in Japan J Clin Microbiol 2000381331ndash1338

Summer JW Nicholson WL Massung RF PCR ampli-fication and comparison of nucleotide sequences fromthe groESL heat shock operon of Ehrlichia species J ClinMicrobiol 1997 362087ndash2092

Uhaa IJ Maclean JD Greene CR et al A case of humanehrlichiosis acquired in Mali clinical and laboratoryfindings Am J Trop Med Hyg 1992 46161ndash164

Walker DH Dumler JS Emergence of the ehrlichiosis ashuman health problems 1996 46161ndash164

Walls JJ Caturegli P Bakken JS et al Improved sensi-tivity of PCR for diagnosis of human granulocytic Ehrli-chiosis using epank1 genes of Ehrlichia phagocytophila-group Ehrlichiae J Clin Microbiol 2000 38354ndash356

Yu XJ Walker DH Sequence and characterization of anEhrlichia chaffeensis gene encoding 314 amino acidshighly homologous to the NAD A enzyme FEMS Mi-crobiol Let 1997 15453ndash58

Address reprint requests toJoon-Seok Chae DVM PhDCollege of Veterinary MedicineChonbuk National University

Jeonju Jeonbuk 561-756 South Korea

E-mail jschaechonbukackr

KIM ET AL26

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3 Alan Harrison Kevin J Bown Ivan G Horak 2011 Detection of Anaplasma bovis In An UndescribedTick Species Collected from the Eastern Rock Sengi Elephantulus myurus Journal of Parasitology 9761012-1016 [CrossRef]

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5 Sungjin Ko Heung-Chul Kim Young-Cheol Yang Sung-Tae Chong Allen L Richards William J SamesTerry A Klein Jun-Gu Kang Joon-Seok Chae 2011 Detection of Rickettsia felis and Rickettsia typhiand Seasonal Prevalence of Fleas Collected from Small Mammals at Gyeonggi Province in the Republicof Korea Vector-Borne and Zoonotic Diseases 119 1243-1251 [Abstract] [Full Text HTML] [Full TextPDF] [Full Text PDF with Links]

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7 V A Rar T I Epikhina N N Livanova V V Panov E K Doroschenko N M Pukhovskaya N PVysochina L I Ivanov 2011 The study of heterogeneity of 16S rRNA gene and groESL operone inDNA samples of Anaplasma phagocytophilum Ehrlichia muris and ldquoCandidatus Neoehrlichia mikurensisrdquodetermined in Ixodes persulcatus ticks on the territory of Ural Siberia and Far East of Russia MolecularGenetics Microbiology and Virology 262 66-73 [CrossRef]

8 S Harrus A Perlman-Avrahami K Y Mumcuoglu D Morick O Eyal G Baneth 2011 Moleculardetection of Ehrlichia canis Anaplasma bovis Anaplasma platys Candidatus Midichloria mitochondrii andBabesia canis vogeli in ticks from Israel Clinical Microbiology and Infection 173 459-463 [CrossRef]

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10 Kaoru Yoshimoto Yuuki Matsuyama Hironori Matsuda Leo Sakamoto Kotaro Matsumoto NaoakiYokoyama Hisashi Inokuma 2010 Detection of Anaplasma bovis and Anaplasma phagocytophilum DNAfrom Haemaphysalis megaspinosa in Hokkaido Japan Veterinary Parasitology 1681-2 170-172 [CrossRef]

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17 V RAR N LIVANOVA V PANOV I KOZLOVA N PUKHOVSKAYA N VYSOCHINA S TKACHEVL IVANOV 2008 Prevalence of Anaplasma and Ehrlichia species in Ixodes persulcatus ticks and smallmammals from different regions of the Asian part of Russia International Journal of Medical Microbiology298 222-230 [CrossRef]

18 M OOSHIRO S ZAKIMI Y MATSUKAWA Y KATAGIRI H INOKUMA 2008 Detection ofAnaplasma bovis and Anaplasma phagocytophilum from cattle on Yonaguni Island Okinawa JapanVeterinary Parasitology 1543-4 360-364 [CrossRef]

19 Do-Hyeon Yu Ying-Hua Li Ji-Seon Yoon Jong-Hyeon Lee Mi-Jin Lee Il-Jeoung Yu Joon-Seok ChaeJin-Ho Park 2008 Ehrlichia chaffeensis Infection in Dogs in South Korea Vector-Borne and ZoonoticDiseases 83 355-358 [Abstract] [Full Text PDF] [Full Text PDF with Links]

20 Joon-Seok Chae Jennifer Zipser Adjemian Heung-Chul Kim Sungjin Ko Terry A Klein Janet Foley2008 Predicting the Emergence of Tick-Borne Infections Based on Climatic Changes in Korea Vector-Borne and Zoonotic Diseases 82 265-276 [Abstract] [Full Text PDF] [Full Text PDF with Links]

21 Joon-Seok Chae Do-Hyeon Yu Smriti Shringi Terry A Klein Heung-Chul Kim Sung-Tae Chong In-Yong Lee Janet Foley 2008 Microbial pathogens in ticks rodents and a shrew in northern Gyeonggi-donear the DMZ Korea Journal of Veterinary Science 93 285 [CrossRef]

22 Takashi Tsunoda 2007 Seasonal Change in Habitat Use by Haemaphysalis longicornis (Acari Ixodidae)Plant Litter and Soil Journal of Medical Entomology 444 575-579 [CrossRef]

23 Hisashi InokumaVectors and Reservoir Hosts of Anaplasmataceae 199-212 [CrossRef]24 B CHAHAN Z JIAN X XUAN Y SATO H KABEYA K TUCHIYA K ITAMOTO M OKUDA T

MIKAMI S MARUYAMA 2005 Serological evidence of infection of and in domestic animals in XinjiangUygur Autonomous Region area China Veterinary Parasitology 1343-4 273-278 [CrossRef]

25 PJ Kelly AC Midwinter J-M Rolain D Raoult 2005 Polymerase chain reaction (PCR) survey forrickettsias and bartonellas in ticks from New Zealand New Zealand Veterinary Journal 536 468-469[CrossRef]

ties now accept that these three bacteria com-prise a single species (Dumler et al 2001 Sum-ner et al 1997) Thus the E phagocytophilagroup as well as Ehrlichia bovis and Ehlrichiaplatys are now classified within the genusAnaplasma because of their close relationship toAnaplasma marginale by phylogenetic studiesusing the 16S rRNA gene and groESL (Dumleret al 2001 Fuente et al 2001)Anaplasmataceae are transmitted to humans

and animals by specific species of ticks fromwild-animal reservoirs (Barlough et al 1997abBreitschwerdt et al 1998 Cao et al 2000 Lizet al 2000 Pusterla et al 1999b Pusterla et al1999c) E chaffeensis has been found in Ambly-omma americanum (Lone star tick) A testudi-narium Haemaphysalis yeni and H flava (Cao etal 2000) A phagocytophilum has been identi-fied not only in Ixodes scapularis and Ixodes paci-ficus ticks but also in Ixodes ricinus (sheep tick)(Barlough et al 1997a Pusterla et al 1999aSchouls et al 1999) The diagnosis of diseasescaused by these bacteria is based on clinicalsigns laboratory findings tick infestation sea-son and geographical location (Barlough et al1996 Barlough et al 1997a Rikihisa 1991Walker et al 1996) Laboratory detection meth-ods include PCR amplification of ehrlichialDNA in blood of suspected hosts or vectors(Breitschwerdt et al 1998 Georges et al 2001Massung et al 1998) PCR makes it possible toidentify the presence of bacterial DNA in cul-ture-negative samples from patients with sus-pected infection (Pusterla et al 2000) Com-petitive PCR is a standard method for thequantification of DNA and has been used suc-cessfully in a number of studies However thistechnique is labor intensive and requires re-sults from multiple reactions to be analyzed foreach sample The causative agents of ehrli-chiosis in humans and animals were detectedfrom many kinds of ticks around the worldbut they have not yet been identified in KoreaHeo et al (2002) reported that detection of Echaffeensis and A phagocytophilum antibody inhuman sera by IFA test and Western im-munoblot blot analysis Therefore we col-lected 799 longicornis three I persulcatus andone I turdus ticks from nine provinces of Ko-rea to identify Ehrlichia and Anaplasma speciesby TaqMan PCR followed by in-depth exami-

nation of TaqMan PCR-positive DNA samplesfor A phagocytophilum and E chaffeensis by PCRmethods and sequencing

MATERIALS AND METHODS

Tick collection

Ticks were collected by dragging a flannelcloth over vegetation or by removing attachedticks on host animals such as cattle horsesgoats dogs cats hedgehogs and wild miceTicks were identified to the species level andto developmental stage A total of 1467 ticksmdash1463 longicornis three I Persulcatus and one Iturdusmdashwere collected from nine provinces(Gangwon Gyeonggi Chungbuk ChungnamGyeongbuk Gyeongnam Jeonbuk Jeonnamand Jeju) of Korea A total of 102 adult femaleticks collected from Jeju province were usedto obtain salivary gland and midgut They werestored in 15 mL Eppendorf tubes at 220degC in-dividually until DNA extraction was per-formed

DNA extraction

Each individual adult and nymph tick (total664) or 139 pools of larvae (5ndash10 ticks per pool)in Eppendorf tubes was mechanically homog-enized using sterile scissors 102 additionaladult female ticks were used to obtain one ofthe two salivary glands and the midgut for PCRamplification DNA was extracted with the QIAamp tissue kit (Qiagen Valencia CA) ac-cording to the manufacturerrsquos instructions

TaqMan real-time and conventional polymerasechain reaction

The approach applied for characterization ofEhrlichia and Anaplasma species was to screenall ticks and selected tick tissues with the useof a highly sensitive real time polymerase chainreaction (PCR) based upon the TaqMan systemfor the presence of 16S rRNA genes of Anaplas-mataceae Tick samples that were positive in thissensitive but broadly reactive TaqMan assaywere subjected to analysis by PCR to identifythe presence of either E chaffeensis (nadA PCR)or A phagocytohilum (nest PCR) Some ampli-

KIM ET AL18


TABLE1

RESU

LTS

OFTAQM

ANREAL-T

IMEPCR F

OR

THED

ETECTIO

NOFEHRLICHIA

ANDANAPLASMA

SPP I

NW

HOLETIC

KS

ANDTHEIR

ORGANS

(SALIV

ARYG

LAND

ANDM

IDGUT) C

OLLECTED

FROM

KOREA


Adult females

Larval

Adult

Whole

Salivary

Unclassified

Provinces

poolsa

Nymphs

males

ticks

gland

Midgut

ticks

Total

Gan

gwon

00

01

10 (10

)0

00

110

(10

)Gye

ongg

i75

122

(61

5)

48c 111 (432)

66b

93 (71

)53

b 85

(62

4)

00

024

241

1 (589)

Chu

ngbu

k0

621 (28

6)

24 (50)

215

(13

3)

00

010

40

(25

)Chu

ngna

m2

10 (20

)0

00

00

02

10 (20

)Gye

ongb

uk

00

07

20 (35

)0

00

720

(35

)Gye

ongn

am3

3 (100

)15

20 (75

)2

2 (100

)0

00

020

25

(80

)Jeon

buk

04 (0)

19 (111)

07

20 (35

)0

08

63 (12

7)

1696

(16

7)

Jeon

nam

08

10 (80

)0

310

(30

)0

00

12 (0)

1132

(34

4)

Jeju

00

01120

(55

)18

75

(24

)19

27

(70

4)

737

(18

9)

5515

9 (346)

Total

801

39 (57

6)

78171 (456)

7099

(70

7)

8418

0 (467)

1875

(24

)19

27

(70

4)

1511

2 (134)

364

803 (453)

a 5ndash10 tick

s per poo

lbInclud

ing on


c Inc

luding on

e Ixodes turdus







KIM ET AL20









RESULTS






Adult females








KIM ET AL22



DISCUSSION







Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85




KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26




























TABLE1

RESU

LTS

OFTAQM

ANREAL-T

IMEPCR F

OR

THED

ETECTIO

NOFEHRLICHIA

ANDANAPLASMA

SPP I

NW

HOLETIC

KS

ANDTHEIR

ORGANS

(SALIV

ARYG

LAND

ANDM

IDGUT) C

OLLECTED

FROM

KOREA


Adult females

Larval

Adult

Whole

Salivary

Unclassified

Provinces

poolsa

Nymphs

males

ticks

gland

Midgut

ticks

Total

Gan

gwon

00

01

10 (10

)0

00

110

(10

)Gye

ongg

i75

122

(61

5)

48c 111 (432)

66b

93 (71

)53

b 85

(62

4)

00

024

241

1 (589)

Chu

ngbu

k0

621 (28

6)

24 (50)

215

(13

3)

00

010

40

(25

)Chu

ngna

m2

10 (20

)0

00

00

02

10 (20

)Gye

ongb

uk

00

07

20 (35

)0

00

720

(35

)Gye

ongn

am3

3 (100

)15

20 (75

)2

2 (100

)0

00

020

25

(80

)Jeon

buk

04 (0)

19 (111)

07

20 (35

)0

08

63 (12

7)

1696

(16

7)

Jeon

nam

08

10 (80

)0

310

(30

)0

00

12 (0)

1132

(34

4)

Jeju

00

01120

(55

)18

75

(24

)19

27

(70

4)

737

(18

9)

5515

9 (346)

Total

801

39 (57

6)

78171 (456)

7099

(70

7)

8418

0 (467)

1875

(24

)19

27

(70

4)

1511

2 (134)

364

803 (453)

a 5ndash10 tick

s per poo

lbInclud

ing on


c Inc

luding on

e Ixodes turdus







KIM ET AL20









RESULTS






Adult females








KIM ET AL22



DISCUSSION







Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85




KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26

































KIM ET AL20









RESULTS






Adult females








KIM ET AL22



DISCUSSION







Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85




KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26

































RESULTS






Adult females








KIM ET AL22



DISCUSSION







Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85




KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26































KIM ET AL22



DISCUSSION







Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85




KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26




























DISCUSSION







Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 977 989 998 998 994 997 981 957 924 916 921 922 9652 21 981 976 976 972 975 967 951 912 905 912 91 9883 10 18 989 989 985 988 971 953 917 91 915 913 9694 2 22 10 100 996 999 979 958 924 916 921 921 9645 2 22 10 0 996 999 979 956 924 916 921 921 9646 6 26 13 4 4 995 975 956 922 913 919 919 967 3 23 11 1 1 5 978 957 923 915 92 92 9638 18 31 27 19 19 23 20 959 913 912 911 915 9689 39 45 44 38 38 41 39 38 913 913 913 908 953

10 71 82 77 71 71 73 72 81 81 971 98 969 90911 78 88 84 78 78 81 79 82 81 27 966 956 9112 73 82 79 73 73 75 74 83 81 19 32 965 90713 73 84 81 73 73 75 74 79 85 29 41 32 90814 32 11 29 33 33 37 34 30 43 85 84 86 85




KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26





























KIM ET AL24








Anaplasma bovis





ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26































ACKNOWLEDGMENTS



REFERENCES

















































KIM ET AL26





























































KIM ET AL26

































































Documents

Identification of Ehrlichia chaffeensis , Anaplasma phagocytophilum , and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus Ticks from Korea