Granulocytic Ehrlichiae in Ixodes persulcatus Ticks from an Area in China Where Lyme Disease Is Endemic

Abstract

A total of 372 adult Ixodes persulcatus ticks were collected from vegetation in a forest area of Heilongjiang Province in northeastern China, where Lyme disease is known to be endemic. The ticks were examined for the presence of granulocytic ehrlichiae by heminested PCR with primers derived from the 16S rRNA gene. Of 310 ticks obtained from the Dahe forestry farm, two pools (each containing 5 ticks) were found positive, with a minimum infection rate of 0.6%. Ehrlichial DNA was also detected in one female (1.6%) of 62 ticks collected from the Yulin forestry farm. The overall minimum infection rate of the 372 I. persulcatus adults was 0.8%. The nucleotide sequences of 919-bp PCR products from the three positive tick specimens were identical to each other and very closely related to the members of the Ehrlichia phagocytophila genogroup. This is the first identification of granulocytic ehrlichiae in ticks in Asia and the first report of infection in I. persulcatus anywhere.

Human granulocytic ehrlichiosis (HGE) was initially described in the United States in 1994 (8) and presents clinically as an acute febrile illness characterized by fever, headache, chills, myalgia, lethargy, and arthralgia (28). Laboratory findings suggestive of the disease mainly include leukopenia, anemia, thrombocytopenia, and elevated hepatic aminotransferase levels (2, 28). The causative agent of HGE has not yet been fully defined, but 16S rRNA gene sequence analysis demonstrates that it is closely related to Ehrlichia equi, the agent of the worldwide equine granulocytic ehrlichiosis, and Ehrlichia phagocytophila, the well-recognized pathogen of tick-borne fever of ruminants in Europe (8, 28). In addition, the HGE agent can cause a form of granulocytic ehrlichiosis in horses (4, 14, 15) and dogs (10). It is suggested that the HGE agent, E. equi, and E. phagocytophila may constitute variants of a single species, now called the E. phagocytophila genogroup.

The granulocytic ehrlichiae have been associated with ixodid ticks that may act as vectors, including Ixodes scapularis (16, 19) and Ixodes pacificus (5, 12, 25) in the United States and Ixodes ricinus (11, 22, 27) in Europe. These ticks are known to transmit Borrelia burgdorferi, the pathogen of Lyme disease, and recent studies have indicated that they could be coinfected with B. burgdorferi and granulocytic ehrlichiae (7, 13, 25). It is suggested that the natural cycle of granulocytic ehrlichiae is probably similar to that of B. burgdorferi (28). Ixodes persulcatus is the vector of Lyme borreliosis (1) and tick-borne encephalitis in northeastern China, but the occurrence of Ehrlichia in ticks has not been established for this region. The objectives of this study were to determine whether or not ehrlichial DNA is present in I. persulcatus ticks in an area where Lyme disease is endemic and to provide initial data regarding the presence of granulocytic ehrlichiae in China.

MATERIALS AND METHODS

Tick collection.

Adult I. persulcatus ticks were collected from a forest area of Heilongjiang Province in northeastern China in 1997. The collection site is near Mudanjiag located at 50° N latitude and 128° E longitude, which is a highland city (elevation from 500 to 600 m above sea level) in the Small Xing-An Mountains. In the area investigated, I. persulcatus is abundant, and Lyme borreliosis is known to be endemic, as indicated by a report of human infection (1) and isolation of B. burgdorferi from ticks (26). In this study, ticks were collected by dragging a standard 1-m² flannel flag over vegetation and were stored alive in the refrigerator until use.

Processing of tick specimens.

Ticks were processed individually or in pools (each containing five ticks). DNA extraction was performed by a modification of a method previously described (16). Briefly, the ticks were placed into micro-tubes and mechanically crushed with sterile scissors in 50 μl of DNA extraction buffer (10 mM Tris [pH 8.0], 2 mM EDTA, 0.1% sodium dodecyl sulfate, 500 μg of proteinase K per ml). The samples were incubated for 2 h at 56°C and then boiled at 100°C for 10 min to inactivate the proteinase K. After centrifugation, the supernatant was transferred to fresh sterile microtubes and purified by two extractions with an equal volume of phenol-chloroform. The DNA was precipitated by adding 3 volumes of ice-cold absolute ethanol and 100 μl of 3 M sodium acetate to the samples and placing them at −20°C for 24 h. The DNA was pelleted at 10,000 × g for 15 min at 4°C in a microcentrifuge and washed twice with ice-cold 70% ethanol. After being dryed, the DNA was resuspended in 50 μl of DNase-free water and used as a template for PCR amplification.

Heminested PCR amplification.

Heminested PCR amplifications were performed with primers designed to amplify the 16S rRNA gene of the E. phagocytophila genogroup. Primers GE9f (5′-AACGGATTATTCTTTATAGCTTGCT-3′) and GE10r (5′-TTCCGTTAAGAAGGATCTAATCTCC-3′), previously described by Chen et al. (8), were applied for the initial amplification. Two primer pairs were used in the heminested PCR amplification. The primer pair GE9f and GE2 (5′-GGCAGTATTAAAAGCAGCTCCAGG-3′) (17) can specifically produce a 546-bp fragment, and the primer pair Ehr521 (5′-TGTAGGCGGTTCGGTAAGTTAAAG-3′) (19) and GE10r yields a 441-bp product. The primary reaction used 3 μl of purified DNA as the template in a total volume of 30 μl. The heminested PCR was performed with 1 μl of the primary PCR product as the template in a volume of 30 μl. For either initial or nested amplification, the reaction mixture contained 200 μM each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 0.8 U of Taq polymerase, and 0.5 μM each primer. The PCR amplification was performed in a Perkin-Elmer 480 thermal cycler. The cycling conditions were identical for primary and nested amplifications, which involved the following step-wise procedure: preheating at 95°C for 2 min; 35 cycles of 94°C for 1 min, 55°C for 75 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. Reaction products were then analyzed by agarose gel electrophoresis or purified for DNA sequencing. A negative control (distilled water) and a positive control (a plasmid containing the 16S rRNA gene of the HGE agent [GenBank accession no. U02521]) were included with each set of amplifications. To minimize contamination, DNA extraction, the reagent setup, amplification, and agarose gel electrophoresis were performed in separate rooms.

Cloning of PCR products and DNA sequencing.

PCR products after nested amplification were purified and then ligated into the plasmid vector pGEM-T (Promega Corp.) according to the manufacturer's instructions. The ligation products were transformed into Escherichia coli XL1-Blue, and white colonies were selected after growth on Luria-Bertani agar with IPTG (isopropyl-β-d-thiogalactopyranoside), X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), and ampicillin. For sequence analyses, recombinant plasmids were extracted and purified from overnight cultures by using the QIA prep Spin Miniprep kit (QIAGEN). The nucleotide sequence of the plasmid insert was determined by a dideoxynucleotide cycle sequencing method with an automated fluorescent ABI PRISM 377 DNA sequencer (Perkin-Elmer, Inc.).

Nucleotide sequence accession number.

The nucleotide sequence reported in this study has been deposited in GenBank under accession no. AF205140.

RESULTS

A total of 372 adult I. persulcatus ticks were examined for the presence of granulocytic ehrlichiae by heminested PCR with initial primer pair GE9f and GE10r and subsequent primer pair GE9f and GE2. The prevalences of PCR-positive ticks from the different sources and by sex are shown in Table 1. The results are expressed as the positive rate or the minimum positive rate, obtained by dividing the positive number of specimens by the total number of ticks examined. The calculation is based on the assumption that each PCR-positive pool contains at least one tick with detectable ehrlichiae. Ticks collected from the Dahe forestry farm were examined in pools, each containing five ticks. Two of 62 pools (310 ticks) were found positive, and the minimum infection rate was 0.6%. Of 62 ticks from the Yulin forestry farm, ehrlichial DNA was detected in one female, with a positive rate of 1.6%. Overall, the minimum positive rate of the 372 ticks was estimated as 0.8%. When the tick samples were examined with the initial primer set of GE9f and GE10r and subsequent primer set of Ehr521 and GE10r, exactly the same results were obtained.

TABLE 1.

Results of heminested PCR for the identification of granulocytic ehrlichiae in I. persulcatus ticks with initial primer pair GE9f and GE10r and subsequent primer pair GE9f and GE2

Origin	Sex	No. of ticks tested	No. positive	Positive rate (%)
Dahe	Male	120 (24 pools)	1 pool	0.8a
	Female	190 (38 pools)	1 pool	0.5a
Yulin	Male	23	0	0
	Female	39	1	2.6
Total		372	3	0.8a

^aMinimum positive rate. 

The primary PCR product (amplified with primer GE9f and GE10r) of each positive specimen was respectively reamplified with subsequent primer sets GE9f-GE2 and Ehr521-GE10r. For all nested PCR amplicons, both DNA strands were sequenced twice. As a result, a specific nucleotide sequence 919 bp long was obtained for each tick specimen. The ehrlichial 16S rDNA sequences determined from the three positive tick samples were identical to each other and all differed from the corresponding sequences of the HGE agent, E. phagocytophila, and E. equi by 4 bases, respectively, but at different positions (Table 2). A variable region was discovered near the 5′ end of 16S rRNA gene at positions 76 to 84 (according to the HGE agent [GenBank accession no. U02521]). It is remarkable that the G at position 77 or 80 was unique to the Ehrlichia variant in I. persulcatus. In contrast, there is an A at both positions for all known granulocytic ehrlichia variants (data not shown).

TABLE 2.

Nucleotide differences among the 919-bp partial 16S rRNA gene sequences of the Ehrlichia variant in I. persulcatus and members of the E. phagocytophila genogroup

Organism	Nucleotide difference at positiona					GenBank accession no.
	76	77	80	84	886
HGE agent	A	A	A	G	G	U02521
E. equi	A	A	A	A	—b	M73223
E. phagocytophila	A	A	A	A	—	M73220
Ehrlichia variant in I. persulcatus	G	G	G	A	G	AF205140

^aPosition of the nucleotide relative to the sequence of the HGE agent reported by Chen et al. (8). 

^b—, no nucleotide corresponds to the HGE agent. A gap was required at this position to align the adjacent sequences. 

DISCUSSION

Granulocytic Ehrlichia DNA was amplified from I. persulcatus collected in a forest area of Heilongjiang Province in northeastern China, where Lyme disease is known to be highly endemic. To our knowledge, this is the first detection of granulocytic ehrlichiae in ticks in Asia and the first report of infection in I. persulcatus anywhere. I. scapularis. and I. pacificus have been identified as potential vectors of the HGE agent and E. equi in the United States (3, 6, 12, 23). The E. phagocytophila genogroup has been found in I. ricinus ticks from many European countries (11, 22, 27). The findings of this study add to the evidence that the E. phagocytophila genogroup is specifically associated with the I. persulcatus complex. I. persulcatus ticks are distributed over an extensive area from Russia to eastern Asia, where about one-fifth of the human population of the world resides. The presence of granulocytic ehrlichiae in northeastern China suggests a potential health threat to both humans and animals in the area, where I. persulcatus ticks are abundant. Detailed epidemiological studies are required to investigate the distribution of ticks infected with ehrlichiae, to determine the animal reservoirs of the ehrlichial agents, and especially to detect ehrlichiae in patients with acute febrile illnesses following tick bite in areas where Lyme disease is endemic.

The overall minimum infection rate of granulocytic ehrlichiae in I. persulcatus adults in this study was 0.8%, which is comparable to that in adult I. pacificus ticks from California (5) and in free-living adult I. ricinus ticks from areas in Switzerland where tick-borne fever is endemic (21). The percentage reported in this study might have been falsely reduced, because ticks were examined in pools, and some pools might contain more than one positive tick. However, even if every tick in the positive pools had harbored detectable ehrlichiae, the overall prevalence would have only gone up to 3.0%. A higher prevalence of the E. phagocytophila genogroup was reported in I. scapularis in the United States (16, 19, 25) and I. ricinus in some European countries (11, 24, 27). This discrepancy in the rates of positivity could be due to differences in sampling approaches, tick species, and ehrlichial life cycle; to geographic and seasonal variations among infected ticks; or to limits of PCR sensitivity.

Sequence analysis of PCR products from tick samples revealed a granulocytic ehrlichia variant that slightly differs from members of the E. phagocytophila genogroup (Table 2). The sequence variants of the granulocytic ehrlichia 16S rRNA gene have previously been detected in ticks in many places (7, 17, 20). It is unlikely that the 16S ribosomal DNA variants represent different Ehrlichia species. The specific sequence polymorphism of the Ehrlichia variant identified in this study has not been reported before, and whether the variant can cause disease in humans and animals remains to be determined.

I. persulcatus is abundant in northeastern China and is known as the vector of B. burgdorferi, the agent of Lyme disease (1). Studies elsewhere have demonstrated that ixodid ticks are coinfected with B. burgdorferi and the HGE agent ((7, 13, 25), and simultaneous human infection with the two agents has been reported (9, 18, 29). Coinfection may explain variations in clinical manifestations in humans and animals as a consequence of tick bites. The identification of granulocytic ehrlichiae in I. persulcatus suggests the probability of coinfection and cotransmission of the two agents in the area. Further studies are needed to investigate this phenomenon, and the possible occurrence of ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite in northeastern China, particularly when clinical symptoms and signs are atypical for Lyme disease.