Asian Pacific Journal of Tropical Medicine (2012)459-464 459 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document heading doi: Investigation of Borrelia spp. in ticks (Acari: Ixodidae) at the border crossings between China and Russia in Heilongjiang Province, China Shi Liu1, Chao Yuan2, Yun-Fu Cui1*, Bai-Xiang Li3, Li-Jie Wu3, Ying Liu4 1The 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, People s Republic of China 2 ' Daqing Oilfield General Hospital Group Rangbei Hospital, Daqing, 163114, People s Republic of China 3 ' Harbin Medical University School of Public Health, Harbin 150001, People s Republic of China 4 ' The 3rd Affiliated Hospital of Qiqihar Medical College, QiqiHar 161002, People s Republic of China ' ARTICLE INFO ABSTRACT Article history: Objective: Borrelia To investigate the precise species of tick vector andMethods: the spirochete pathogen Received 15 February 2012 at the Heilongjiang Province international border with Russia. In this study, ticks were Received in revised form 15 March 2012 collected from 12 Heilongjiang border crossings (including grasslands, shrublands, forests, and Accepted 15 April 2012 plantantions) to determine the rate and species type of spirochete-infected ticks and the most Available online 20 June 2012 Results: prevalent spirochete genotypes. The ticks represented three genera and four species Ixodes persulcatus Dermacentor silvarum Haemaphysalis concinna of the Ixodidae family [ , , and Haemaphysalis japonica Ixodes persulcatus Borrelia burgdorferi sensu ]. had the highest amount of Keywords: lato Borrelia Ixodes persulcatus infection of 25.6% and the most common species of isolated from Borrelia garinii Conclusions: Borrelia garinii Lyme disease was , strain PD91. Our results suggest that PD91- Tick Ixodes persulcatus Borrelia burgdorferi sensu lato infected may be the principal cause of Lyme disease in the border crossing Borrelia garinii areas of Heilongjiang Province. clinically reported by at least 29 provinces, and more than 1. Introduction Borrelia burgdorferi 130 strains of sensu lato have been isolated from infected patients, ticks, and animals[5,6]. Lyme disease is a tick-borne zoonotic infection caused The prevalence of Lyme disease is known to correlate Borrelia by spirochetal bacteria of the genus[1]. Three with the geographic distributions and activities of vector Borrelia Ixodes scapularis species of account for the majority of cases in ticks[7]. In North America, the (Acari: Borrelia burgdorferi sensu stricto Ixodidae dentatus North America and Asia ( ) Ixodidae) and (Acari: Ixodidae) are Borrelia afzelii B. afzelii Borrelia garinii and Europe [ ( ) and the most common vectors in the Eastern regions[8], while B. garinii Ixodidae pacificus ( )][2]. The annual global incidence was estimated (Acari: Ixodidae) is the most common in Ixodidae ricinus at more than 0.3 million cases in 2009[3], with a projected the western regions[9]. In Europe, (Acari: Borrelia increase in case number due to continued urban sprawl and Ixodidae) is the most common vector[10]. In China, burgdorferi Ixodidae increased deforestation and contact between humans and sensu lato has been isolated from persulcatus I. persulcatus Ixodidae animals. ( )(Acari: Ixodidae), granulatus I. granulatus Ixodidae In China, the first reported case of Lyme disease came ( ) (Acari: Ixodidae), acutitarsus Haemaphysalis concinna H. from the forest region of Hailin County in Heilongjiang (Acari: Ixodidae), ( concinna Haemaphysalis japonica H. Province in 1986[4]. Since then, Lyme disease has been ) (Acari: Ixodidae), ( japonica Haemaphysalis longicornis H. ) (Acari: Ixodidae), ( longicornis Haemaphysalis bispinosa ) (Acari: Ixodidae), * H. bispinosa Haemaphysalis cornigera : Corresponding author: Prof. Yun-Fu Cui, Department of General Surgery, The ( ) (Acari: Ixodidae), Rhipicephalus microplus 2nd Affiliated Hospital of Harbin Medical University, No.148 Baojian Road, Nangang (Acari: Ixodidae), (Acari: District, Harbin 150001, People's Republic of China. Dermacentor nuttalli Ixodidae), (Acari: Ixodidae), and Tel: +86-451-86605113 Dermacentor silvarum D. silvarum E-mail: [email protected] ( ) (Acari: Ixodidae)[11,12]. et al./Asian Pacific Journal of Tropical Medicine (2012)459-464 460 Shi Liu Each of these species occupies different geographic niches. I. persulcatus The species is the most prevalent tick in the Inner Monglia, Heilongjiang, Jilin, Liaoning, Xinjiang, H. longicornis Ningxia and Hebei Provinces. dominates the I. granulatus Henan and Shandong Provinces. dominates the Fujian, Guangdong, Yunnan and Guizhou Provinces. H. bispinosa is most prevalent in Sichuan, Hunan, Hubei, Jiangxi, Jiangsu and Anhui Provinces[13]. The Heilongjiang Province in Northeast China is not only an endemic region of Lyme disease but represents a unique envirionment that supports transmission since it is situated on the Russian border and the site of international trade. However, little is known about the distinctive epidemiologic factors of the 12 border crossings encompassed within this region. Thus, this study was designed to investigate the Borellia rates and species of spirochete-infected ticks and pathogens in the 12 border crossings of Heilongjiang. Figure 1. Survey areas. ° ′ ° ′ ° ′ ° ′ 1, Dongning (43 25 -44 35 N and 129 53 -131 18 E); 2, Suifenhe 2. Materials and methods ° ′ ° ′ ° ′ ° ′ ° ′ (43 20 -44 40 N and 130 00 -130 40 E); 3, Mishan (45 01 - ° ′ ° ′ ° ′ ° ′ ° ′ 45 55 N and 131 14 -133 08 E); 4, Hulin (45 23 -46 36 N and ° ′ ° ′ ° ′ ° ′ ° ′ 2.1. Survey area and tick collection 132 11 -133 56 E); 5, Raohe (46 30 -47 34 N and 133 07 - ° ′ º ′ º ′ º ′ º ′ 134 20 E); 6, Tongjiang (47 25 -48 17 N and 132 18 -134 07 E); ° ′ ° ′ ° ′ ° ′ 7, Luobei (47 12 -48 21 N and 130 01 -131 34 E); 8, Fuyuan ° ′ ° ′ ° ′ ° ′ º ′ The 12 Heilongjiang border crossings include Dongning, (47 25 -48 27 N and 133 40 -135 05 E); 9, Jiayin (48 83 - º ′ º ′ º ′ ° ′ ° ′ Suifenhe, Mishan, Hulin, Raohe, Fuyuan, Tongjiang, Luobei, 49 26 N and 129 09 -130 50 E); 10, Xunke (47 58 -49 36 N ° ′ ° ′ º ′ º ′ º ′ Jiayin, Xunke, Heihe, and Mohe. These crossings join China to and 127 24 -129 17 E); 11, Heihe (47 42 -51 03 N and 124 45 - ° ´ ° ´ º ′ ° ′ ° ′ ° ′ ° ′ the far-Eastern region of Russia, located at 43 25 -50 47 N and 129 18 E); and 12, Mohe (52 10 -53 33 N and 121 07 -124 20 E). ° ´ ° ´ 125 03 135 05 1 - E (Figure ). The international border region 2.3. DNA extraction ranges in altitude from 40 m to 1 100 m above sea level, and has a subarctic climate (3.6 曟 annual mean temperature, ~120 day frost-free season, and 500-600 mm annual mean rainfall) and Total DNA was extracted from the cultured spirochetes, geographic regions ranging from swamps and wetlands to plains a panel of international standard strains (B31, 20047 and VS461 PD91, and mountains. ), and a panel of Chinese representative strains ( FP1, GS1 CS4 For this study, ticks were collected by flagging from and ; supplied by the Epidemiology Institute, plantations, forests, shrublands and grasslands of each of the Chinese Center for Disease Control, Beijing, China) using the previously described method[16]. DNA concentration was 12 border crossing areas during their peak activity period determined by spectrophotometry. (early May to mid June) in 2009. All specimens were stored at 4 曟 until analyses. 2.4. PCR and sequencing 2.2. Spirochete isolation and cultivation PCR amplification of the 5S-23S rRNA gene from each subculture of spriochetes was carried out by the previously An entomologist (Lin Shi) identified the species of the described method[17]. PCR products were separated using collected ticks. Isolation and cultivation of spirochetes from 1.5% agarose gel electrophoresis, stained with ethidium infected ticks were carried out according to the previously bromide, visualized under UV, and gel purified. An aliquot described methods[14]. The spirochetes from inidividual ticks of the purified sample was confirmed by sequencing (Boya were cultivated for eight weeks, with weekly monitoring Biotechnology Corp., Shanghai, China). by dark-field microscopy, after which the cultures split 2.5. Restriction fragment length polymorphism (RFLP) and propagated as subcultures. The infectivity rate of analysis each spirochete culture was determined according to the previously described procedure[15]. Once identified, the strains were deposited in the repository at the Heilongjiang For RFLP anlaysis, 5S-23S rRNA amplicons were digested Mse Dra Province Center for Disease Control. with 栺 and 栺, as previously described[18]. The et al./Asian Pacific Journal of Tropical Medicine (2012)459-464 Shi Liu 461 digested DNA was then electrophoresed through 16% 60 polyacrylamide gels for 3 h at 100 V. The gels were silver 50 stained and the banding pattern was visualized under white 40 light. 30 2.6. Statistical analysis 20 Spirochete prevalence ratios prevalence Spirochete Chi 10 The -square test was used to evaluate the preferred 0 habitats among the various tick species. One-way ANOVA Hulin Jiayin Mohe Chi Mishan Raohe Fuyuan Luobei Xunke and -square tests were used to examine the regional DongningSuifenhe Tongjiang differences for the various tick species. The spirochete Figure 2. I. persulcatus prevalence ratios were calculated as binomial error The spirochete prevalence ratios of at different Chi locations. estimates and analyzed among the locations by the - ± ± P From left to right, the ratios (%)