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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anaplasma phagocytophila is transmitted by genus Ixodes ticks, including I. scapularis,6,7 and I. pacificus8,9 in the United States, I. ricinus in Europe,10,11 and I. persulcatus in Asia.12 These ticks also serve as the vectors of Borrelia burgdorferi, the causative agent of Lyme disease. Coinfection of ticks with A. phagocytophila and B. burgdorferi has been increasingly reported in recent years.13–16 Northeastern China is highly endemic for Lyme disease,17 where the infection rate of B. burgdorferi in adult I. persulcatus ticks can be as high as 44.1%.18 Recently, the presence of A. phagocytophila in ticks from the region has been reported.12 Therefore, an extensive study was carried out to confirm the previous findings, and to investigate coinfection of I. persulcatus ticks with A. phagocytophila and B. Burgdorferi.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Extraction of DNA. After identification, ticks were soaked in 70% ethanol for a few minutes, and then rinsed three times in sterile water. Extraction of DNA was performed as previously described.19 Briefly, the ticks were placed into microtubes and mechanically disrupted 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/mL). The samples were incubated for two hours at 56°C and then boiled at 100°C for 10 minutes to inactivate the proteinase K. After centrifugation, the supernatant was transferred to fresh sterile microtubes and purified by extracting twice with an equal volume of phenol-chloroform before use.
Amplification by a polymerase chain reaction (PCR). A nested PCR was performed with primers designed to amplify the 16S rRNA gene of A. phagocytophila.12 Primers GE9f and GE10r, previously described by Chen and others,4 were used for the primary amplification. The PCR amplifications were performed in a volume of 30 µL in a Perkin-Elmer (Norwalk, CT) model 2400 thermal cycler. An initial three-minute denaturation at 95°C was followed by 35 cycles at 94°C for 15 seconds, 55°C for 20 seconds, and 72°C for 15 seconds, and a final extension at 72°C for five minutes. In nested PCR, the components and conditions were similar to those for the primary amplification, except that primers GE9f and GE2 were used,20 and 1 µL of the primary PCR product was used as the template. A positive control (a plasmid containing the 16S rRNA gene of the HGE agent (kindly provided by Dr. J. Stephen Dumler, Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD) and a negative control (distilled water) were included with each set of amplifications.
For amplification of B. burgdorferi DNA, a nested PCR was carried out with primers derived from the B. burgdorferi outer surface protein A (ospA) gene.21 For the primary amplification, 3 µL of each template sample was amplified in a 30-µL reaction mixture containing the primers OA1 and OA4. One microliter of the primary PCR product was then used as the template in a second 30-µL reaction mixture with primers BSL and OA4. The PCR amplifications were performed in a Perkin-Elmer model 2400 thermal cycler using the following protocol: preheating at 95°C for three minutes, followed by 40 cycles at 94°C for 20 seconds, 50°C for 20 seconds, and 72°C for 30 seconds, and a final extension at 72°C for seven minutes. In parallel with each amplification of tick specimens, DNA from a B. burgdorferi isolate was used as a positive control and distilled water was used as a negative control.
Reaction products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. 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. The products of nested amplification were collected, and the purified DNA fragments were then cloned into the plasmid vector pGEM-T (Promega Corp., Madison, WI) and transformed into competent cells (Escherichia coli XL1-Blue) according to the manufacturer’s instructions. The recombinant plasmids were extracted and purified from overnight cultures using QIA prep Spin Miniprep Kit (Qiagen, Valencia, CA). The nucleotide sequences of the plasmid inserts were determined by a dideoxynucleotide cycle sequencing method with an automated DNA sequencer (ABI PRISM 377; Perkin-Elmer).
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RESULTS |
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. Anaplasma phagocytophila were detected in 4.6% of the ticks. Of 643 I. persulcatus ticks collected from the Wuerqihan and Moerdaoga forestry farms in the Great Xingan Mountains of Inner Mongolia, 40 (6.2%) contained A. phagocytophila. The difference in infection rates among male, female, and nymphal ticks was not significant (
2 = 1.69, degrees of freedom [df] = 2, P = 0.43). The frequency of positive ticks from the Weihe forestry farm in the Small Xingan Mountains of Heilongjiang was 3.1% (22 of 702 ticks), which was significantly lower than that at Inner Mongolia (2 = 6.59, df = 1, P = 0.01). Although A. phagocytophila DNA was not detected in nymphal ticks in this area, the frequency of positive ticks was not statistically different regardless of sex or stage (2 = 3.71, df = 2, P = 0.16).
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2 = 29.28, df = 1, P < 0.001). In contrast to infection with A. phagocytophila, infection with B. burgdorferi was more prevalent in ticks in the Small Xingan Mountains (39.3%) than that in the Great Xingan Mountains (2 = 19.80, df = 1, P < 0.001). There was a significant difference in B. burgdorferi infection between adult and nymphal ticks from the Small Xingan Mountains. (2 = 8.95, df = 1, P = 0.003). Among 62 A. phagocytophila-positive I. persulcatus ticks, seven were coinfected with B. burgdorferi. The overall coinfection rate was 0.5%. No difference in proportions of coinfected ticks was demonstrated between the two studied areas.
Ten ticks positive for A. phagocytophila by the nested PCR were randomly selected for sequence analysis. Of the 10 positive specimens, six (3 males, 2 females, and 1 nymph) were from the Great Xingan Mountains and four (2 males and 2 females) were from the Small Xingan Mountains. A 919-basepair nucleotide sequence amplified with primer pair GE9f and GE10r4 was obtained from each tick specimen. Sequence analysis of the 10 PCR amplicons revealed three variants of A. phagocytophila (Table 2). The nucleotide sequences from two male and one female ticks from the Great Xingan Mountains and one male tick from the Small Xingan Mountains were identical to the published sequences of the HGE agent. In addition, two sequence variants were detected. Variant 1, isolated from a male, a female, and a nymphal tick from the Great Xingan Mountains, had a T instead of an A at position 81 according to the corresponding sequence of the HGE agent (GenBank accession no. U02521). Variant 2, isolated from a male and two female ticks from the Small Xingan Mountains, differed from the HGE agent sequence by four bases at position 76, 77, 80, and 84 (Table 2).
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DISCUSSION |
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Ixodes persulcatus ticks are distributed over an extensive area from Russia to eastern Asia, where approximately one-fifth of the human population of the world resides. The results obtained in the present survey demonstrate that A. phagocytophila infection poses a potential health threat to both humans and animals where I. persulcatus is abundant, and should be useful in alerting public health officials and clinicians about the presence of ehrlichiosis in northeastern China. In contrast to our study, an investigation conducted in the Baltic region of Russia failed to demonstrate A. phagocytophila infection in I. persulcatus.25 In southern Germany, ehrlichia-positive ticks were only found in one of five surveyed regions, arguing that the distribution of A. phagocytophila seems to be focal.26 Further epidemiologic studies are required to clarify the diversity of A. phagocytophila, to identify its natural foci, and especially to define potential human and animal infection risks following tick bites in areas infested with I. persulcatus.
In forest areas of northeastern China, I. persulcatus is the most abundant tick species and is responsible for the majority of tick bites in humans. This tick species infests multiple animal hosts, and therefore, may acquire more than one pathogen from different reservoirs. Coinfection of ixodid ticks with A. phagocytophila and B. burgdorferi has been reported in the United States and many European countries with various prevalences.9,10,15,16,24 In our study, seven of 62 ehrlichia-positive ticks were found to harbor both A. phagocytophila and B. burgdorferi. Coexistence of the two pathogens in I. persulcatus ticks from Asia has not been previously reported. This finding suggests that humans may become coinfected with the two pathogens as a consequence of a single tick bite. In fact, simultaneous human infection with the two agents has already been reported,27 and it may lead to variations in clinical symptoms and signs.28,29
If one considers that A. phagocytophila can cause immunosuppression in its mammalian hosts, coinfection with two or more tick-borne agents may aggravate the clinical pictures of Lyme disease and tick-borne encephalitis. The identification of A. phagocytophila in I. persulcatus, and the finding of coinfection with B. burgdorferi in the current study imply that the possible occurrence of ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite in the forest areas of northeastern China, particularly when clinical manifestations are atypical for Lyme disease. Unfortunately, the B. burgdorferi genospecies was not determined in the present investigation. A previous study reported 64.4% B. garinii and 35.6% B. afzelii based on a PCR-restriction fragment length polymorphism analysis of 45 B. burgdorferi isolates from this area.18 Whether the coinfection was associated with B. burgdorferi genospecies remains to be determined.
The nucleotide sequences of PCR products from tick samples were all identified as part of A. phagocytophila 16S rRNA gene, and showed high level of identity (99.6–100%) with published sequences of the HGE agent (Table 2
). Three types of A. phagocytophila sequences were found: one with the identical sequence of the HGE agent, and two variants with nucleotide differences from the HGE agent by one and four basepairs, respectively. A variable region was found near the 5' end of 16S rRNA gene at the position from 76 to 84 (according to HGE agent [GenBank accession no. U02521]). This result is consistent with findings of previous studies in other places, in which nucleotide differences were also identified in this variable region but at different positions.13,20,23,30,31 It remains to be determined whether each molecular variant of A. phagocytophila can cause a disease in humans or animals.
Received June 20, 2002. Accepted for publication January 3, 2003.
Acknowledgments: We are grateful to Dr. Rong-man Xu for identifying ticks and reading the manuscript, and to Dr. Jian-hua Zhu and Dr. Shan-hu Chen (The Central Anti-Epidemic Station of the Great Xingan Mountains) for assistance in collection of ticks.
Financial support: This study was supported by the National Natural Science Foundation of China (grant no. 39970655) and the Beijing Natural Science Foundation (grant no. 7992029).
Authors’ addresses: Wu-Chun Cao, Qiu-Min Zhao, Pan-He Zhang, Hong Yang, Xiao-Ming Wu, Bo-Hai Wen, and Xi-Tan Zhang, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da-Jie Street, Fengtai District, Beijing 100071, People’s Republic of China. J. Dik F. Habbema, Center for Decision Sciences in Tropical Disease Control, Department of Public Health, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Reprint requests: Dr. Wu-Chun Cao, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da-Jie Street, Fengtai District, Beijing 100071, People’s Republic of China, Telephone/Fax: 86-10-63812060, E-mail: caowc{at}nic.bmi.ac.cn
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REFERENCES |
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  S. J. Swanson, D. Neitzel, K. D. Reed, and E. A. Belongia Coinfections Acquired from Ixodes Ticks Clin. Microbiol. Rev., October 1, 2006; 19(4): 708 - 727. [Abstract] [Full Text] [PDF]
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 W.-C. CAO, L. ZHAN, J. HE, J. E. FOLEY, S. J. DE VLAS, X.-M. WU, H. YANG, JAN. H. RICHARDUS, and J. D. F. HABBEMA NATURAL ANAPLASMA PHAGOCYTOPHILUM INFECTION OF TICKS AND RODENTS FROM A FOREST AREA OF JILIN PROVINCE, CHINA. Am J Trop Med Hyg, October 1, 2006; 75(4): 664 - 668. [Abstract] [Full Text] [PDF]
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