SHORT COMMUNICATIONS

Molecular Detection of Ehrlichia chaffeensisand Anaplasma bovis in the Salivary Glands

from Haemaphysalis longicornis Ticks

Mi-Jin Lee1 and Joon-Seok Chae2

Abstract

The salivary gland (SG) of tick plays an important role as a route in the dissemination of tick-borne pathogens totheir hosts. We evaluated the presence of these pathogens in the SGs of Haemaphysalis longicornis ticks, and theseticks were collected from grazing cattle in Jeju Island, Korea. Of total 463 one-side SGs, 56 (12.1%) SGs werepositive for Ehrlichia chaffeensis and 11 (2.4%) were positive for Anaplasma bovis. In addition, two (0.4%) SGs wereco-infected with both E. chaffeensis and A. bovis. In conclusion, we specifically describe the presence ofE. chaffeensis and A. bovis in the SGs of H. longicornis ticks in Korea.

Key Words: Anaplasma bovis—Ehrlichia chaffeensis—Haemaphysalis longicornis—Salivary gland—Tick.

Human monocytic ehrlichiosis and human granulo-cytic anaplasmosis are tick-borne, emerging zoonotic

diseases caused by Ehrlichia chaffeensis and Anaplasma phago-cytophilum, respectively. These pathogens are transmittedfrom ticks to their hosts through the bite of ticks, and thesalivary gland (SG) of ticks is a major route in the dissemi-nation of these tick-borne pathogens (Sauer et al. 2000).

Economic loss caused by tick-borne diseases has been re-ported in ruminants, including other domestic animalsthroughout the world. Both Ehrlichia and Anaplasma spp. weredetected in Haemaphysalis longicornis ticks in Korea and Japan(Kim et al. 2003, 2006, Kawahara et al. 2006, Chae et al. 2008).Especially, E. chaffeensis has been detected in whole H. long-icornis ticks collected from various animals such as cattle,horse, dogs, and rodents in Korea (Lee et al. 2005). However,these pathogens have not yet been detected from the SG ofH. longicornis tick, although the SG plays an important rolein the dissemination of tick-borne diseases. Thus, we con-ducted this study to evaluate the presence of E. chaffeensis andA. phagocytophilum in the SGs of H. longicornis ticks fromgrazing cattle in Jeju Island, Korea, because the H. longicornistick causes large economic loss in grazing cattle in Jeju Island.

Ticks were collected from grazing cattle in Jeju Island,Korea. After identification of tick morphology by microscopy,we selected only 463 adult female H. longicornis ticks. The SGswere obtained by separating from the other internal organs

and tick exoskeleton. Each SG was rinsed with sterile salinethree times and stored individually in each microcentrifugetubes at�808C until needed. To operate the polymerase chainreaction (PCR) assay, genomic DNA was extracted from a sideof each SG.

At first, to identify the infection of E. chaffeensis 16S rRNAgene in the SGs, the nested PCR assay was conducted, and weused primers and PCR conditions designed by Murphy et al.(1998). Primers ECC and ECB were used to amplify all Ehrli-chia spp., and then primers HE1 and HE3 were used for theE. chaffeensis–specific amplification. Each 20mL PCR mixturecontained 5 mL of template DNA, 2mL of 10�PCR buffer in-cluding 20 mM MgCl2, 1 mL of a 10 mM dNTPs mixture, 3mLof each primer (1 pmol=mL), and 0.2 mL of 5 U=mL Taq DNApolymerase (Intron Biotechnology, Daejeon, Korea). Next, thenested PCR assay for A. phagocytophilum 16S rRNA gene wasconducted using primers and PCR conditions designed byBarlough et al. (1996). Primers EE1 and EE2 were used for theprimary PCR assay, and primers EE3 and EE4 were used forthe secondary PCR assay. Each 25mL PCR mixture contained5mL of template DNA, 2.5 mL of 10�PCR buffer including20 mM MgCl2, 1 mL of a 10 mM dNTPs mixture, 3mL of eachprimer (1 pmol=mL), and 0.15 mL of 5 U=mL Taq DNA poly-merase (Intron Biotechnology). Additionally, we used H.longicornis ticks that are bred in normal rabbit without tick-borne diseases as a negative control whenever PCR reaction is

1Laboratory of Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Korea.2Laboratory of Internal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, Korea.

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performed. All PCR products were then separated by 1.0%agarose gel electrophoresis, stained with ethidium bromide,and photographed using a Gel-Doc 2000 system (Bio-Rad,Hercules, CA). The amplified PCR products were cloned toconfirm the nucleotides sequence. Sequence homology sear-ches were conducted using the National Center for Bio-technology Information (NCBI, National Institute of Health,MD) Basic Local Alignment Search Tool (BLAST) networkservice. The nucleotide sequences were then aligned andcompared using the MultAlin software (Multiple sequencealignment by Florence Corpet) and homology searches usinga GeneStream Align (Genestream network service; IGH,Montpellier, France).

In the results of the nested PCR assay, 56 (12.1%) of 463 SGswere positive for E. chaffeensis when an approximately 390 bpamplicon band was observed. Eleven (2.4%) SGs were posi-tive for A. phagocytophilum when an amplicon of approxima-tely 926 bp was observed. However, sequencing of the 926 bpamplicon and subsequent analysis indicated the presence ofAnaplasma bovis rather than A. phagocytophilum. In addition,two (0.4%) SGs were positive for both E. chaffeensis and A.bovis (Table 1). Sequence analysis for E. chaffeensis and A. bovis16S rRNA gene obtained from this study was accomplishedthrough GenBank of the National Center for BiotechnologyInformation (National Institute of Health) BLAST networkservice, and they were deposited into GenBank accessionnumbers EU181141 (SG24), EU181140 (SG32), EU181144(SG38), EU181142 (SG175), and EU181143 (SG176), respec-tively.

In sequence analysis of three SGs (SG24, SG32, and SG38)positive for E. chaffeensis, their homology is very high with oneanother. Of those, the sequence of SG24 was 99.7%, 99.7%, and98.2% homologous with that of the United States (GenBankaccession number, GAN AF416764), Korea (GAN AY350424),and China (GAN AF414399), respectively. In addition, thesequences of SG32 and SG38 were 97.7% and 98.7% homolo-gous with those of the United States (GAN AF416764), Korea(GAN AY350424), and China (GAN AF147752), respectively.Sequence analysis of A. phagocytophilum was accomplished fortwo SGs (SG175 and SG176), and these two nucleotide se-quences were verified as A. bovis that is very close to A. pha-gocytophilum in sequence homology. The homology betweenA. bovis and A. phagocytophilum was 99.2%. The sequence ofSG175 was 98.9%, 98.8%, and 98.4% homologous with that ofKorea (GAN AF470698), Japan (GAN AB196475), and SouthAfrica (GAN U03775), respectively. Also, the sequence ofSG176 was 99.2%, 99.0%, and 98.8% homologous with that ofJapan (GAN AB196475), Korea (GAN AF470698), and SouthAfrica (GAN U03775), respectively.

In this short note, we have reported the presence ofE. chaffeensis and A. bovis in H. longicornis ticks and animals inKorea. The infection of E. chaffeensis was first reported in anactive-duty American soldier stationed in Korea (Sachar2000), and antibodies against E. chaffeensis were identified inKorean patients with febrile illness (Heo et al. 2003). In ad-dition, the presence of E. chaffeensis was reported from H.longicornis ticks, Apodemus agrarius, and various animals inKorea (Kim et al. 2003, 2006; Lee et al. 2005, 2009, Chae et al.2008). Further, A. bovis were detected from H. longicornis ticksand a deer in Korea, respectively (Kim et al. 2003, Lee et al.2009). However, though SG play an important role in trans-mission of tick-borne diseases, the confirmation of E. chaf-feensis and=or A. bovis infection in the SG of tick has not beenreported in Korea yet. Therefore, we describe for the first timethe natural infection of E. chaffeensis and A. bovis in the SGs ofH. longicornis ticks in Korea.

Jeju Island has warm climate during April to October. Awarming trend species such as H. longicornis tick can easilyinfect grazing cattle with tick-borne diseases during thesemonths within Jeju Island. Despite the use of acaricides tocontrol tick populations, the high prevalence of these tick-borne diseases presents a major constraint to improvementof livestock production. Both E. chaffeensis and A. bovis residein monocytes of blood and cause various clinical sings inhosts. Therefore, the presence of E. chaffeensis and A. bovis inthe SGs of ticks parasitized especially cattle reveals thatthese tick-borne pathogens impact health of the cattlepopulation in Korea. Further, both E. chaffeensis (humanmonocytotropic ehrlichiosis) and A. bovis (ruminant mono-cytotropic anaplasmosis) have to be considered as epidemi-ological important pathogens in humans and other animalspecies in Korea.

Disclosure Statement

This work was supported by Research Settlement Fund forthe new faculty of Seoul National University (SNU).

References

Barlough, JE, Madigan, JE, DeRock, E, et al. Nested polymerasechain reaction for detection of Ehrlichia equi genomic DNA inhorses and ticks (Ixodes pacificus). Vet Parasitol 1996; 63:319–329.

Chae, JS, Yu do, H, Shringi, S, et al. Microbial pathogens in ticks,rodents and a shrew in northern Gyeonggi-do near the DMZ,Korea. J Vet Sci 2008; 9:285–293.

Heo, EJ, Park, J, Koo, JR, et al. Serologic and molecular detectionof Ehrlichia chaffeensis and Anaplasma phagocytophila (humangranulocytic ehrlichiosis agent) in Korean patients. J ClinMicrobiol 2003; 40:3082–3085.

Kawahara, M, Rikihisa, Y, Lin, Q, et al. Novel genetic variants ofAnaplasma phagocytophilum, Anaplasma bovis, Anaplasma cen-trale, and a novel Ehrlichia sp. in wild deer and ticks on twomajor islands in Japan. Appl Environ Microbiol 2006; 72:1102–1109.

Kim, CM, Kim, MS, Park, MS, et al. Identification of Ehrlichiachaffeensis, Anaplasma phagocytophilum, and A. bovis in Haema-physalis longicornis and Ixodes persulcatus ticks from Korea.Vector Borne Zoonot Dis 2003; 3:17–26.

Kim, CM, Yi, YH, Yu, DH, et al. Tick-borne rickettsial pathogensin ticks and small mammals in Korea. Appl Environ Microbiol2006; 72:5766–5776.

Table 1. Polymerase Chain Reaction Results

for the Infection of Ehrlichia chaffeensis

and Anaplasma bovis in the One-Side Salivary

Glands of Haemaphysalis longicornis Ticks

Collected from Grazing Cattle in Jeju Island, Korea

Number of infected salivary glands (%)

Number of testedsalivary glands

Ehrlichiachaffeensis

Anaplasmabovis Co-infection

463 56 (12.1) 11 (2.4) 2 (0.4)

412 LEE AND CHAE

Lee, SO, Na, DK, Kim, CM, et al. Identification and prevalence ofEhrlichia chaffeensis infection in Haemaphysalis longicornis ticksfrom Korea by PCR, sequencing and phylogenetic analysisbased on 16S rRNA gene. J Vet Sci 2005; 6:151–155.

Lee, MJ, Yu, DH, Yoon, JS, et al. Natural co-infection of Ehrlichiachaffeensis and Anaplasma bovis in a deer in South Korea. J VetMed Sci 2009; 71:101–103.

Murphy, GL, Ewing, SA, Whttworth, LC, et al. A molecular andserologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingiiin dogs and ticks from Oklahoma. Vet Parasitol 1998; 79:325–339.

Sachar, DS. Ehrlichia chaffeensis infection in an active duty soldierstationed in Korea. MSMR 2000; 6:9–11.

Sauer, JR, Essenberg, RC, Bowman, AS. Salivary glands in ixo-did ticks: control and mechanism of secretion. J Insect Physiol2000; 46:1069–1078.

Address correspondence to:Joon-Seok Chae, DVM, Ph.D.Veterinary Internal Medicine

College of Veterinary MedicineSeoul National University

Seoul 151-742Korea

E-mail: jschae@snu.ac.kr

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