Molecular Detection of Ehrlichia chaffeensis and Anaplasma bovis in the Salivary Glands from Haemaphysalis longicornis Ticks

H uman monocytic ehrlichiosis and human granulocytic anaplasmosis are tick-borne, emerging zoonotic diseases caused by Ehrlichia chaffeensis and Anaplasma phagocytophilum, respectively. These pathogens are transmitted from ticks to their hosts through the bite of ticks, and the salivary gland (SG) of ticks is a major route in the dissemination of these tick-borne pathogens (Sauer et al. 2000).

Economic loss caused by tick-borne diseases has been reported in ruminants, including other domestic animals throughout the world. Both Ehrlichia and Anaplasma spp. were detected 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. longicornis 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 of H. longicornis tick, although the SG plays an important role in the dissemination of tick-borne diseases. Thus, we conducted this study to evaluate the presence of E. chaffeensis and A. phagocytophilum in the SGs of H. longicornis ticks from grazing cattle in Jeju Island, Korea, because the H. longicornis tick 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 SGs were obtained by separating from the other internal organs and tick exoskeleton. Each SG was rinsed with sterile saline three times and stored individually in each microcentrifuge tubes at −80°C until needed. To operate the polymerase chain reaction (PCR) assay, genomic DNA was extracted from a side of each SG.

At first, to identify the infection of E. chaffeensis 16S rRNA gene in the SGs, the nested PCR assay was conducted, and we used primers and PCR conditions designed by Murphy et al. (1998). Primers ECC and ECB were used to amplify all Ehrlichia spp., and then primers HE1 and HE3 were used for the E. chaffeensis–specific amplification. Each 20 μL PCR mixture contained 5 μL of template DNA, 2 μL of 10 × PCR buffer including 20 mM MgCl₂, 1 μL of a 10 mM dNTPs mixture, 3 μL of each primer (1 pmol/μL), and 0.2 μL of 5 U/μL Taq DNA polymerase (Intron Biotechnology, Daejeon, Korea). Next, the nested PCR assay for A. phagocytophilum 16S rRNA gene was conducted using primers and PCR conditions designed by Barlough et al. (1996). Primers EE1 and EE2 were used for the primary PCR assay, and primers EE3 and EE4 were used for the secondary PCR assay. Each 25 μL PCR mixture contained 5 μL of template DNA, 2.5 μL of 10 × PCR buffer including 20 mM MgCl₂, 1 μL of a 10 mM dNTPs mixture, 3 μL of each primer (1 pmol/μL), and 0.15 μL of 5 U/μL Taq DNA polymerase (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 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 to confirm the nucleotides sequence. Sequence homology searches were conducted using the National Center for Biotechnology Information (NCBI, National Institute of Health, MD) Basic Local Alignment Search Tool (BLAST) network service. The nucleotide sequences were then aligned and compared using the MultAlin software (Multiple sequence alignment by Florence Corpet) and homology searches using a GeneStream Align (Genestream network service; IGH, Montpellier, France).

In the results of the nested PCR assay, 56 (12.1%) of 463 SGs were positive for E. chaffeensis when an approximately 390 bp amplicon band was observed. Eleven (2.4%) SGs were positive for A. phagocytophilum when an amplicon of approximately 926 bp was observed. However, sequencing of the 926 bp amplicon and subsequent analysis indicated the presence of Anaplasma 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. bovis 16S rRNA gene obtained from this study was accomplished through GenBank of the National Center for Biotechnology Information (National Institute of Health) BLAST network service, and they were deposited into GenBank accession numbers EU181141 (SG24), EU181140 (SG32), EU181144 (SG38), EU181142 (SG175), and EU181143 (SG176), respectively.

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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 tested salivary glands	Ehrlichia chaffeensis	Anaplasma bovis	Co-infection
463	56 (12.1)	11 (2.4)	2 (0.4)

In sequence analysis of three SGs (SG24, SG32, and SG38) positive for E. chaffeensis, their homology is very high with one another. Of those, the sequence of SG24 was 99.7%, 99.7%, and 98.2% homologous with that of the United States (GenBank accession number, GAN AF416764), Korea (GAN AY350424), and China (GAN AF414399), respectively. In addition, the sequences of SG32 and SG38 were 97.7% and 98.7% homologous with those of the United States (GAN AF416764), Korea (GAN AY350424), and China (GAN AF147752), respectively. Sequence analysis of A. phagocytophilum was accomplished for two SGs (SG175 and SG176), and these two nucleotide sequences were verified as A. bovis that is very close to A. phagocytophilum in sequence homology. The homology between A. bovis and A. phagocytophilum was 99.2%. The sequence of SG175 was 98.9%, 98.8%, and 98.4% homologous with that of Korea (GAN AF470698), Japan (GAN AB196475), and South Africa (GAN U03775), respectively. Also, the sequence of SG176 was 99.2%, 99.0%, and 98.8% homologous with that of Japan (GAN AB196475), Korea (GAN AF470698), and South Africa (GAN U03775), respectively.

In this short note, we have reported the presence of E. chaffeensis and A. bovis in H. longicornis ticks and animals in Korea. The infection of E. chaffeensis was first reported in an active-duty American soldier stationed in Korea (Sachar 2000), and antibodies against E. chaffeensis were identified in Korean patients with febrile illness (Heo et al. 2003). In addition, the presence of E. chaffeensis was reported from H. longicornis ticks, Apodemus agrarius, and various animals in Korea (Kim et al. 2003, 2006; Lee et al. 2005, 2009, Chae et al. 2008). Further, A. bovis were detected from H. longicornis ticks and a deer in Korea, respectively (Kim et al. 2003, Lee et al. 2009). However, though SG play an important role in transmission of tick-borne diseases, the confirmation of E. chaffeensis and/or A. bovis infection in the SG of tick has not been reported in Korea yet. Therefore, we describe for the first time the natural infection of E. chaffeensis and A. bovis in the SGs of H. longicornis ticks in Korea.

Jeju Island has warm climate during April to October. A warming trend species such as H. longicornis tick can easily infect grazing cattle with tick-borne diseases during these months within Jeju Island. Despite the use of acaricides to control tick populations, the high prevalence of these tick-borne diseases presents a major constraint to improvement of livestock production. Both E. chaffeensis and A. bovis reside in monocytes of blood and cause various clinical sings in hosts. Therefore, the presence of E. chaffeensis and A. bovis in the SGs of ticks parasitized especially cattle reveals that these tick-borne pathogens impact health of the cattle population in Korea. Further, both E. chaffeensis (human monocytotropic ehrlichiosis) and A. bovis (ruminant monocytotropic anaplasmosis) have to be considered as epidemiological important pathogens in humans and other animal species in Korea.