SHORT COMMUNICATIONS

Molecular Detection of Bartonella grahamiiand B. schoenbuchensis-Related Species in Korean

Water Deer (Hydropotes inermis argyropus)

Sungjin Ko,1 Su-jin Kim,1 Jun-gu Kang,1 Sohyun Won,1 Hang Lee,2 Nam-shik Shin,3 Kyoung–seong Choi,4

Hwa-young Youn,1 and Joon-seok Chae1

Abstract

We determined the prevalence of Bartonella spp. and investigated which species of Bartonella naturally infects Koreanwater deer (KWD, Hydropotes inermis argyropus) in the Republic of Korea (ROK). A total of 70 spleens from KWDcarcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife (CGRB) in the ROKbetween 2008 and 2009. Nested PCRs were performed using the rpoB gene and internal transcribed spacer (ITS)region primers to amplify the DNA fragment of Bartonella. Using ITS-based nested PCR, Bartonella grahamii andBartonella schoenbuchensis–related species were detected in 11 (15.8%) and 9 (12.9%) of 70 KWD spleens, respectively.The 11 B. grahamii amplicons were classified into 2 genotypes by sequence analysis. Using rpoB-based nested PCR, B.grahamii was detected in 5 (7.1%) of 70 KWD spleen samples. This is the first report of B. grahamii and B. schoen-buchensis in KWD, suggesting that KWD may act as reservoirs for the spreading of Bartonella spp. in the ROK.

Key Words: Korean water deer—Hydropotes inermis argyropus—Bartonella grahamii—Bartonella schoenbuchensis.

Introduction

The bacteria Bartonella, which are Gram-negative, facul-tative, obligate, intracellular alphaproteobacteria, can in-

fect erythrocytes and endothelial cells of various hosts andcause several human diseases, including cat scratch disease,lymphadenitis, and peliosis hepatitis (Dehio 2005). Wide-spread occurrence and diversity of the genus Bartonella havebeen reported increasingly in recent years. With efficientmolecular methods for detection now available, the numberof new species is increasing rapidly, and over the last fewyears, new zoonotic species have been demonstrated.

In the Republic of Korea (ROK), Bartonella spp. have beendetected in wild rodents, ticks, and companion animals (Chaeet al. 2008, Kim et al. 2009), and B. elizabethae was isolated fromwild rodents (Kim et al. 2005). Recently, several human casesof Bartonella infection have been reported (Suh et al. 2010,Yoon et al. 2010).

Water deer (Hydropotes inermis) are indigenous to the lowerreaches of rivers in China and the Korean peninsula. They aredivided into 2 subspecies—the Korean water deer (KWD;

Hydropotes inermis argyropus) and the Chinese water deer(Hydropotes inermis inermis). Wild deer are an important res-ervoir of Bartonella spp. infection in the United States andEurope (Dehio et al. 2001, Matsumoto et al. 2008). Further-more, in the ROK, Anaplasma phagocytophilum, A. bovis, andTheileria spp. were detected in wild water deer (Han et al.2009, Kang et al. 2011). These findings indicate that wild deercould be reservoirs for other vector-borne diseases. However,Bartonella spp. have not been evaluated in water deer andother wild deer in Asia.

The aim of the present study was to investigate the prev-alence of Bartonella DNA in KWD and determine whichBartonella species could infect water deer in the ROK. Weused nested PCR and phylogenetic analysis based on theinternal transcribed spacer (ITS) region and rpoB gene toconduct molecular detection and genetic diversity analysis ofBartonella spp. from naturally infecting wild KWD.

KWD carcasses (N = 70) were collected in Chungcheong-buk-do (n = 5), Gangwon-do (n = 16), Gyeonggi-do (n = 11),and Jeollanam-do (n = 18) provinces, Seoul City (n = 4), andUlsan City (n = 16) in the ROK between March, 2008, and

1Laboratory of Veterinary Internal Medicine, 2Conservation Genome Resource Bank for Korean Wildlife, 3Laboratory of Zoo and WildlifeMedicine, Research Institute and BK21 Program for Veterinary Science and College of Veterinary Medicine, Seoul National University, Seoul,Republic of Korea.

4School of Animal Science and Biotechnology, College of Ecology and Environmental Sciences, Kyungpook National University, Sangju,Republic of Korea.

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August, 2009, by the Conservation Genome Resource Bankfor Korean Wildlife (CGRB).

DNA was extracted from spleens using DNeasy Blood andTissue Kits (Qiagen Inc., Valencia, CA) according to themanufacturer’s instructions. Bartonella-specific DNA frag-ments were amplified in 2 different nested PCR reactions withdifferent target genes to confirm the results and to differen-tiate Bartonella species. All samples were screened using 2 setsof species-specific nested PCR primers targeting the ITS re-gion (QHVE OF, 5¢-TTCAGATGATGATCCCAAGC-3¢;QHVE OR, 5¢-AACATGTCTGAATATATCTTC-3¢; QHVE IF,5¢-CCGGAGGGCTTGTAGCTCAG-3¢; QHVE IR, 5¢-CACAATTTCAATAGAAC-3¢) and rpoB gene (BrpoB OF, 5¢-GTAGACTGATTAGAACGCTG-3¢; BrpoB OR, 5¢-CGCATTGGCTTACTTCGTATG-3¢; BrpoB IF, 5¢-GTAGACTGATTAGAACGCTG-3¢; BrpoB IR, 5¢-TTCCCGTACCAACAAATGG-3¢)(Houpikian et al. 2001, Renesto et al. 2001). To avoid nested-PCR contamination, sample preparation, DNA extraction,PCR preparation, and nested-PCR amplification and analysiswere performed in separate rooms with entirely separateequipment, supplies, and solutions. Negative controls, con-sisting of bacteria-free spleen DNA and water control, wereincluded in all the nested PCR runs, and no bands were de-tectable from the negative controls. B. henselae (AmericanType Culture Collection [ATCC] 49882) and B. grahamii(ATCC 700132) genomic DNA were used as positive controlsfor detecting Bartonella species. Amplified products wereseparated by electrophoresis on 1.5% agarose gels, visualizedby ethidium bromide, purified using the QIAquick Gel Ex-traction Kit (Qiagen Inc., Valencia, CA), and sequenced bydideoxy termination with an automatic sequencer (ABI 3730xlcapillary DNA sequencer).

Comparative analyses of the nucleotide sequences werecompleted using Bartonella ITS regions and rpoB gene se-quences in the GenBank database. Phylogenetic analyses ofthe region and rpoB gene fragments were constructed usingthe Clustal X (version 1.60) program and the neighbor-joiningmethod with MEGA 4.0 software. The phylogenetic treebased on the rpoB gene and ITS region shows the positions ofstrains identified in this study (Fig. 1). The sequences obtainedin the present study have been deposited in GenBank, andaccession numbers are shown in Table 1.

The prevalence of Bartonella DNA in KWD was 7.1% (5/70)and 28.6% (20/70) using rpoB gene- and ITS region-detectingnested PCRs, respectively. The homology of 5 amplified rpoBsequences showed 96.9–97.0% similarity to the representativeB. grahamii strain V2 (AF16599). In the ITS region-based PCR,11 B. grahamii (15.8%) and 9 B. schoenbuchensis–related species(12.9%) were identified.

A total of 11 B. grahamii-related sequences were classifiedinto 2 genotypes that showed 91.2% and 100% sequencesimilarity to B. grahamii strain V2 (AJ269785). This resultcorresponds with those of earlier studies that reported thesimilarity of the B. grahamii ITS sequence as 91.9% and 98.8%(Gil et al. 2010). Nine ITS-positive PCR products (sample ID:KWDB 2, 29, 31, 32, 33, 34, 35, 36, and 37) showed low se-quence homology (72.8–74.2%) to the closest relativeB. schoenbuchensis strain R1 (AY116639) and 68.8–70.2% se-quence identity with B. capreoli strain IBS193 (AB498009) (Fig.1B). In phylogenetic trees of the ITS region, those sequencesshowing low sequence similarities (both B. grahamii– andB. schoenbuchensis–related) were grouped independently fromthe closest relative, suggesting that these strains might beconsidered a new species; however, further analysis,

Table 1. Detection of Bartonella grahamii and B. schoenbuchensis rpoB and ITS Partial Gene

from Korean Water Deer Spleens Collected in the Republic of Korea, 2008–2009

rpoB gene ITS region

Sample ID PCR Closest relative (%)a GenBank accession number PCR Closest relative (%)a GenBank accession number

KWDB1 + B. grahamii (97.0%) JN810794 + B. grahamii (91.2%) JN810837KWDB2 - - - + B. schoenbuchensis (74.2%) JN810830KWDB12 + B. grahamii (96.9%) JN810795 + B. grahamii (100%) JN810847KWDB19 + B. grahamii (97.0%) JN810796 + B. grahamii (100%) JN810845KWDB29 - - - + B. schoenbuchensis (74.2%) JN810831KWDB31 - - - + B. schoenbuchensis (74.0%) JN810828KWDB32 - - - + B. schoenbuchensis (72.8%) JN810836KWDB33 - - - + B. schoenbuchensis (74.2%) JN810834KWDB34 - - - + B. schoenbuchensis (74.2%) JN810833KWDB35 - - - + B. schoenbuchensis (74.2%) JN810832KWDB36 - - - + B. schoenbuchensis (74.0%) JN810829KWDB37 - - - + B. schoenbuchensis (74.2%) JN810835KWDB41 - - - + B. grahamii (91.2%) JN810843KWDB46 - - - + B. grahamii (91.2%) JN810838KWDB51 - - - + B. grahamii (91.2%) JN810844KWDB53 - - - + B. grahamii (91.2%) JN810839KWDB58 - - - + B. grahamii (91.2%) JN810842KWDB59 + B. grahamii (97.0%) JN810797 – - -KWDB61 - - - + B. grahamii (91.2%) JN810840KWDB67 - - - + B. grahamii (91.2%) JN810841KWDB69 + B. grahamii (97.0%) JN810798 + B. grahamii (100%) JN810846

aSequence similarity with closest relative [B. grahamii strain V2 (AF16599, AJ269785), B. schoenbuchensis strain R1 (AY116639)].

Bartonella grahamii AND B. schoenbuchensis IN KOREAN WATER DEER 417

including isolation and biochemical and molecular charac-terizations, should be performed to define this genotype as anovel species.

In this study, B. grahamii and B. schoenbuchensis–relatedspecies were identified in the spleens of wild KWD by se-quence analysis. B. schoenbuchensis was first isolated from theblood of wild roe deer (Dehio et al. 2001). Subsequently, thisspecies has been detected mainly in ectoparasites of deer(Matsumoto et al. 2008). Until now, wild rodents were knownas a main reservoir/host of B. grahamii, which is distributedworldwide and detected predominantly in Asian countries,such as China and Japan (Inoue et al. 2009).

This is the first report of the molecular detection of Barto-nella in KWD, suggesting that water deer may serve as amammalian reservoir for transmission of Bartonella spp. Fur-thermore, this result has enabled us to provide additionalinformation on the epidemiology of B. grahamii, the predom-inant Bartonella species in the world.

Several new Bartonella spp. have been identified in wildanimals, and the number of human bartonellosis cases hasbeen rapidly increasing. Our results suggest that further studywill be necessary to determine the relationships and hostspecificity among public health, domestic and wild animals,and Bartonella spp. Furthermore, the impact of these patho-gens on human health, especially in undiagnosed cases offebrile illness, needs to be evaluated.

Acknowledgments

This research was supported by Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science andTechnology (2012-0005524).

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Joon-seok Chae

Veterinary Internal MedicineCollege of Veterinary Medicine

Seoul National UniversitySeoul 151-742

Korea

E-mail: jschae@snu.ac.kr

418 KO ET AL.