- Email: [email protected]
Molecular survey of hard ticks in endemic areas of tick-borne diseases in China
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Molecular survey of hard ticks in endemic areas of tick-borne diseases in China Xin Lu a,1 , Xian-Dan Lin b,1 , Jian-Bo Wang a,1 , Xin-Cheng Qin a , Jun-Hua Tian c , Wen-Ping Guo a , Fei-Neng Fan d , Renfu Shao e , Jianguo Xu a , Yong-Zhen Zhang a,∗ a State Key Laboratory for Infectious Disease Prevention and Control, Department of Zoonoses, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping Liuzi 5, 102206 Beijing, China b Wenzhou Center for Disease Control and Prevention, 325001 Wenzhou, Zhejiang Province, China c Wuhan Center for Disease Control and Prevention, 430022 Wuhan, Hubei Province, China d Cixi Center for Disease Control and Prevention, Cixi 315300, Zhejiang Province, China e GeneCology Research Group, School of Science, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland, Australia
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 22 December 2012 Accepted 9 January 2013 Available online xxx Keywords: Ticks Taxonomy Molecular analysis Phylogeny China
a b s t r a c t Over the past several years, there was a substantial increase in the number of cases of known and novel tick-borne infections in humans in China. To better understand the ticks associated with these infections, we collected hard ticks from animals or around livestock shelters in 29 localities in 5 provinces (Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang) where cases of tick-borne illness were reported. We collected 2950 hard ticks representing 7 species of 4 genera (Dermacentor sinicus, Haemaphysalis flava, Haemaphysalis longicornis, Ixodes granulatus, Ixodes persulcatus, Rhipicephalus microplus, and Rhipicephalus sanguineus). These ticks were identified to species using morphological characters initially. We then sequenced the mitochondrial small subunit rRNA (12S rRNA) gene, cytochrome oxidase subunit 1 (COI) gene, and the second internal transcribed spacer (ITS2) gene of these ticks, and conducted phylogenetic analyses. Our analyses showed that the molecular and morphological data are consistent in the identification of the 7 tick species. Furthermore, all these 7 tick species from China were genetically closely related to the same species or related species found outside China. Rapid and accurate identification and long-term monitoring of these ticks will be of significance to the prevention and control of tick-borne diseases in China. © 2013 Published by Elsevier GmbH.
Introduction Ticks (Acari: Ixodida) are parasitic arachnids with a worldwide distribution. The Ixodida have 3 families, Argasidae (soft ticks), Nuttalliellidae, and Ixodidae (hard ticks), with 907 described species (Bowman and Nuttall, 2008; Guglielmone et al., 2010). Ticks act as reservoirs and vectors of a broad range of pathogens for humans and animals, including viruses, bacteria, rickettsiae, and protozoans (Parola et al., 2005; Swanson et al., 2006; Heyman et al., 2010; Leiby, 2011). These tick-borne pathogens are responsible for more than 100,000 cases of illness in humans each year throughout the world (WHO, 2006). Approximately 10% of the currently recognized tick species are known to carry pathogens of humans and animals (Jongejan and Uilenberg, 2004). Some previously unrecognized or emerging pathogens have been identified recently in ticks, such as Huaiyangshan virus (Zhang et al., 2011) and Ehrlichia species (Pritt et al., 2011). Moreover, zoonotic or vector-borne pathogens
∗ Corresponding author. Tel.: +86 10 58900782; fax: +86 10 58900700. E-mail address: [email protected] (Y.-Z. Zhang). 1 These authors contributed equally to this work and share first authorship.
(especially viruses) show specific association with their hosts (Lovisolo et al., 2003). Traditionally, species identification of ticks has been mainly based on morphological examination. As special skills acquired through extensive experience are needed, only a limited number of experts such as taxonomists and trained technicians can identify ticks to species accurately (Jinbo et al., 2011). Genetic and phylogenetic analyses based on DNA sequences could reveal species groups and assign unknown individuals to species (Uilenberg et al., 2004), and infer the evolutionary relationship of ticks within and between species. Hence, genetic and phylogenetic analyses of molecular markers have been extensively used to identify species and understand phylogenetic relationships of ticks in the past 2 decades (Andrews et al., 1992; Jackson et al., 1998, 2000; Shaw et al., 2002; Song et al., 2011). Several genes, such as the mitochondrial 12S ribosomal RNA gene (12S rRNA), the cytochrome oxidase subunit 1 (CO1), and the second internal transcribed spacer (ITS2), have been used frequently for taxonomical and evolutionary investigation (Barker and Murrell, 2002, 2004; Shao and Barker, 2007; Jinbo et al., 2011). Moreover, DNA (and RNA) extracted from ticks carrying tick-borne diseases collected in endemic areas could be analyzed to identify both etiologic agents and vector tick species as
1877-959X/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
2
well (Marrelli et al., 2007; Zhang et al., 2012). Molecular analyses, thus, are very useful and practical tools for monitoring ticks that are of public health significance. At least 117 species of ticks (by morphological examination) are known to occur in China; these ticks are in 10 genera of 2 families and have a nationwide distribution (Yang et al., 2008; Chen et al., 2010). Cases of well-known tick-borne diseases, such as Lyme disease (Hao et al., 2011), spotted fever (Fan et al., 1987), CrimeanCongo hemorrhagic fever (Sun et al., 2009), and babesiosis (Qi et al., 2011), have been reported from China. In the past several years, there was a substantial increase in the number of the cases of known and novel tick-borne infections in humans in China. Furthermore, the illness occurred in a large area from the north of Inner Mongolia to the south of Hubei province. Recently, human granulocytic anaplasmosis transmitted by ticks was reported from Anhui Province of China (Zhang et al., 2008). Notably, a new hemorrhagic fever-like disease with up to 15% mortality caused by Huaiyangshan virus (HYSV) (a novel tick-borne Bunyavirus) occurred in Henan and Hubei provinces of China in 2009 (Zhang et al., 2011). Further investigation indicated that both Haemaphysalis longicornis and Rhipicephalus microplus can carry HYSV, and the abundance of H. longicornis appears to determine the emergence of the new disease (Zhang et al., 2012). As very few molecular studies on ticks have been conducted in China, most of the 117 species of ticks described from China were identified by morphological examination (Yang et al., 2008; Chen et al., 2010). Thus, far less is known about the phylogenetic relationships between ticks in and outside China. For controlling emerging or reemerging tick-borne diseases and minimizing the impact of them, it is urgent to better understand the molecular data and the phylogenetic relationships of the ticks collected in endemic areas of tick-borne diseases in China. In this study, we identified 7 species of ticks collected from endemic areas of tick-borne diseases in China and conducted molecular phylogenetic analyses among these ticks and other ticks previously identified in China and other parts of the world. Materials and methods Ticks collection In 2010, 2950 adult ticks (males: 882; females: 2068) were collected from animals or around livestock shelters at 29 localities of 5 provinces in China: Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang (Fig. 1). They were collected from hedgehogs (Erinaceus amurensis), sheep (Ovis aries), cattle (Bos taurus), goats (Capra aegagrus hircus), and dogs (Canis lupus familiaris). All ticks were first identified morphologically under light microscope by a trained technician and then verified by molecular analysis.
PCR reactions were performed in 50-l volumes including 2 l DNA (100 ng) and 2.5 U of rTaq DNA polymerase (Takara, Dalian, China). The cycling conditions were: 94 ◦ C for 5 min (initial denaturation) followed by 35 cycles of 94 ◦ C for 30 s (denaturation), 55 ◦ C for 1 min (annealing), 72 ◦ C for 1 min (extension), and a final extension of 72 ◦ C for 10 min. A negative control (no DNA template) was included in each amplification run. Successful amplification was determined by electrophoresing 2 l of the PCR amplicons on 1.5% (w/v) agarose gels (1 × TBE) and visualizing with gold view under UV light. The DL2000 DNA size marker (Takara) was included in the agarose gels. There was only one single band of expected size (approximately 380 bp for the 12S rRNA gene, 660 bp for the COI gene, and 920–1850 bp for the ITS2 gene) in each PCR amplification. PCR amplicons sequencing PCR amplicons were purified using the QIAquick Gel Extraction kit (Qiagen) according to the manufacturer’s recommendations. The purified DNA (<700 bp) was subjected to a direct sequencing. The sequencing primers were the same as the PCR amplification primers. But the purified DNA (>700 bp) was cloned into pMD18-T vector (TaKaRa). Then, the vector was transformed into JM109competent cells. At least 3 DNA clones were used for sequencing by M13 sequencing primers. DNA sequencing was performed using Applied Biosystems 377 gene sequencers by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Automatic DNA sequence assembly was performed by DNA baser software, version 3.2.5. Both the forward and reverse reads were used for sequence assembly; primer sequences were then excluded with SeqMan (DNAstar Inc.). Sequence alignment CLUSTAL X2 software (version 2.0.12) was used to align all sequences obtained in this study and retrieved from GenBank with the multiple alignment modes. For comparing genetic and phylogenetic analysis, all sequences (primer sequences excluded) were kept in the same size after editing with MEGA5. The following dataset sizes were used in the final phylogenetic analysis: 12S rRNA = 72 sequences, 330 nt; COI = 40 sequences, 546 nt; ITS2 = 58 sequences, 852 nt. The nucleotide similarities were calculated using MegAlign (DNAstar Inc.). Calculating maximum likelihood estimate of transition/transversion bias was performed under the Tamura–Nei model with MEGA5 (Tamura et al., 2011). The value of pairwise distance between sequences was calculated in MEGA5 using the Kimura 2parameter model (Kimura, 1980). Standard error estimates were obtained by a bootstrap procedure (1000 replicates). All ambiguous positions were removed for each sequence pair.
PCR amplification of 12S rRNA, COI and ITS2 genes
Phylogenetic analysis
After washing twice with phosphate-buffered saline, ticks were homogenized with a mortar and pestle in 1 ml of phosphatebuffered saline. A total of 200 l of homogenates was used to extract DNA with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. All DNA samples were diluted to 50 ng/l and then subjected to PCR for amplification. Fragments of the 12S rRNA gene, the COI gene and the ITS2 gene were amplified from each tick DNA sample with primer pairs T1B (5 -AAACTAGGATTAGATACCCT-3 ) and T2A (5 -AATGAGAGCGACGGGCGATGT-3 ) (Beati and Keirans, 2001), C1-J-1718 (5 -GGAGGATTTGGAAATTGATTAGTTCC-3 ) and C1-N2329 (5 -ACTGTAAATATATGATGAGCTCA-3 ) (Shao et al., 2001) and TITS2F1 (5 -CGAGACTTGGTGTGAATTGCA-3 ) and TITS2R1 (5 TCCCATACACCACATTTCCCG-3 ) (Chitimia et al., 2009), respectively.
Phylogenetic relationships were estimated with the Bayesian method and the maximum likelihood (ML) method with 1000 replications in the bootstrap test. The Bayesian trees were constructed with Mrbayes v3.1.2 (Huelsenbeck and Ronquist, 2001), and the ML analyses were performed through (Stamatakis http://phylobench.vital-it.ch/raxml-bb/index.php et al., 2008). The model used to construct the trees was chosen by the Modeltest program (version 3.7) (Posada and Crandall, 1998). When trees were constructed based on the 12S rRNA and COI gene sequences, soft tick (Argas species) sequences were used as an outgoup. As the ITS2 sequence of the soft tick was not available in GenBank, the rooted tree of the ITS2 sequences was inferred using the Bayesian MCMC method available in the BEAST v1.6.0 package (Drummond and Rambaut, 2007). Because the MCC tree
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
3
Fig. 1. Locations where ticks were collected in this study. Ticks were collected in Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang provinces. The locations are indicated by triangles.
is automatically rooted on the assumption of a molecular clock, it enables determination of the basal lineage. Accordingly, the basal lineage estimated by the MCC tree was used as the basal lineage to root the phylogenetic trees inferred under the ML phylogenetic analyses. Results Ticks samples In this study, a total of 2950 adult ticks (male: 882; female: 2068) was collected from 5 provinces in China (Table 1, Fig. 1), from where tick-borne diseases (tick-borne encephalitis, Huaiyangshan hemorrhagic fever, Lyme disease, human granulocytic anaplasmosis, and other tick-borne rickettsioses) have been reported (Song et al., 2004; Jiang et al., 2006; Chai et al., 2010; Tian et al., 2010; Zhang et al., 2011). Morphological examination identified these ticks to 7 species of 4 genera of the family Ixodidae: Dermacentor sinicus, Haemaphysalis flava, Haemaphysalis longicornis, Ixodes granulatus, Ixodes persulcatus, Rhipicephalus microplus, and Rhipicephalus sanguineus, respectively. The species, their geographical origins, and GenBank accession numbers of these tick sequences are shown in Figs. 2–4. 12S rRNA gene For all 12S rRNA sequences obtained in China, the sequences varied in length from 337 bp in H. flava to 349 bp in I. persulcatus (excluding the primers). The estimated mean frequencies of the 4 nucleotides were variable for the 7 species as follows: adenine 38.9–43.9%, cytosine 6.6–8.7%, thymidine 33.7–40.5%, and guanine 12–14.8%. The similarities of intraspecific sequences varied from
81% to 100%, while similarities of interspecific sequences within a given genus were estimated from 76.6% to 96.8% (Table 2). The differences of intraspecific sequences among the ticks from different sampling locations were 0–19%, but the differences of intraspecific sequences from the same sampling location were 0–1.9%. In addition, the tick species identified in the present study in China exhibited 0–14.8% differences from the same species or 3.2–23.4% from other species in the same genus from other parts of the world. To investigate the evolutionary relationships between ticks collected in China and around the world, we constructed phylogenetic trees using the 12S rRNA gene sequences obtained in this study and reported previously (retrieved from GenBank) with both Bayesian and ML methods (Fig. 2). A soft tick, Argas reflexus, was used as the outgroup. Consistent with the traditional taxonomy, the ticks collected in this study were grouped into 4 genera, Ixodes, Dermacentor, Haemaphysalis, and Rhipicephalus, with strong support, 0.96, 1.0, 1.0, and 1.0, respectively. All Ixodes ticks were clustered together and formed a phylogroup in both the Bayesian and ML trees, while the ticks from the other 3 genera were grouped into another phylogroup. Within the first phylogroup, as expected, I. persulcatus collected from Inner Mongolia of China was closely related to I. persulcatus (HM234631) from Russia. Furthermore, the species had a closer relationship with I. scapularis (L43901) from the USA, followed by I. ricinus ticks which are distributed widely in Europe. Interestingly, the 3 samples of I. granulatus from Zhejiang Province formed a lineage, and had a closer relationship with I. persulcatus, I. scapularis, and I. ricinus than with I. uriae from Iceland (AM410581). Within the second phylogroup, H. flava and H. longicornis collected from Henan and Hubei provinces of China, where a novel hemorrhagic fever disease transmitted by H. longicornis had been reported in 2009 (Zhang et al., 2011), were closely related to each
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9 4
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Fig. 2. Phylogenetic relationships between ticks found in and outside China. ML/Bayesian trees were based on the 12S rRNA gene sequences. Numbers (>50%/>0.5) above or below branches indicate bootstrap values or posterior node probabilities. Argas reflexus was used as an outgroup. The GenBank accession numbers of the sequences obtained in this study are shown in this figure.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
5
Table 1 The species and geographical locations of tick specimens collected from animal hosts and livestock shelters in this study. Species
Hosts
No. of ticks (male/female) Location
D. sinicus H. flava H. longicornis
I. granulatus I. persulcatus R. microplus
R. sanguineus
Hedgehogs Cattle Dogs Cattle Goats Dogs Livestock shelter Sheep Livestock shelter Cattle Goats Dogs Cattle Goats Livestock shelter
Total
Total
Beijing
Henan
Hubei
Inner Mongolia
Zhejiang
19/37 – – – – – – – – – – – – – –
– – – 79/148 8/18 5/14 – – – 164/329 – – – – –
– 31/58 9/15 42/178 13/42 6/12 – – – 397/936 7/20 2/9 – – –
– – – – – – – 31/72 23/55 – – – – – –
– – – – – – 4/11 – – – – – 25/62 15/41 2/11
19/37 31/58 9/15 121/326 21/60 11/26 4/11 31/72 23/55 561/1265 7/20 2/9 25/62 15/41 2/11
19/37
256/509
507/1270
54/127
46/125
882/2068
other and formed a lineage. These 2 species had a closer relationship (and formed a sub-phylogroup) with Amblyomma species from America and Africa than with ticks of the genera Dermacentor and Rhipicephalus. In the genus Dermacentor, the 4 samples of D. sinicus collected in Beijing clustered together. D. sinicus from China was closely related to D. marginatus from Italy and Switzerland. D. sinicus and D. marginatus had a closer relationship with D. reticulatus (AF150038) from France, and formed a sister taxon of D. andersoni (AF150040) and D. variabilis (AF031851). All Rhipicephalus ticks, including samples of Rhipicephalus species from China and those from other parts of the world, were clustered together and could be divided into 2 lineages. Within the first lineage, R. sanguineus from China was more closely related to R. sanguineus from Thailand (AY987377) and Brazil (EU346676) than those from other parts of the world. Furthermore, R. sanguineus had a close relationship with R. appendiculatus (AF150028) from Uganda. R. microplus ticks collected from Henan and Hubei provinces of China were clustered together and were closely related to R. microplus from other Asian countries, South America, Africa, and Australia. R. microplus had a close relationship with R. kohlsi from Jordan (AF150043), and they formed another lineage with R. pravus and R. kochi from Tanzania.
COI gene The COI sequences obtained from the ticks in China were approximately 610 bp (excluding the primers) in length. The estimated mean frequencies of the 4 nucleotides were as follows: adenine 27.6–31.4%, cytosine 17–19.1%, thymidine 37.4–40.6%, and guanine 13.7–14.9%. The similarities of intraspecific sequences varied from 93.9% to 100%, while similarities of interspecific sequences within genera were estimated from 69.8% to 94.4% (Table 2). The similarities of intraspecific sequences of the ticks from different sampling locations were 93.9–100%, whereas the similarities of intraspecific sequences from the same sampling location were 97.7–100%. In addition, the tick species identified in this study were closely related to the same species (0–6.1% differences), but exhibited 5.6–30.2% differences from other species in the same genus. The phylogenetic relationships among these ticks was inferred with COI gene sequences using the soft tick (A. persicus) as the outgroup (Fig. 3). Consistent with the 12S rRNA trees, the ticks were also divided into 2 phylogroups in the COI trees. The first phylogroup contained only the ticks of the genus Ixodes, while the other 4 genera of ticks formed the second phylogroup. Within the first phylogroup, I. granulatus from Zhejiang Province formed a distinct lineage. I. persulcatus collected from Inner Mongolia of China
Table 2 The average value of pairwise distance and the range of the similarities between sequences of ticks from the same or from different species. Species
Similarities between sequences/(genetic distance between sequences) Within species
D. sinicus H. flava H. longicornis I. granulatus I. persulcatus R. microplus R. sanguineus
Between species
12S rDNA
COI
ITS2
12S rDNA
COI
ITS2
99.5–100.0 0 100 (0–0.003) 99.5–100.0 (0–0.003) 98.6–99.1 (0.004–0.007) 97.9–100.0 0 97.7–100.0 (0–0.028) 81–100.0 (0–0.087)
99.8–100.0 (0–0.002) 99.8–100.0 (0–0.002) 93.9–100.0 (0–0.027) 99.5–99.7 (0.006) 99.7–100.0 (0–0.004) 99.7–100.0 (0–0.003) 100 0
97.7–99.4 0 98.1–100.0 0 89.1–99.4 (0.001–0.017) 89.7–100.0 (0–0.005) 87–97.6 (0–0.009) 89–99.7 (0–0.017) 84.5–100.0 (0–0.009)
83.5–96.8 (0.041–0.166) 91.6–92.1 (0.101–0.109) 91.6–92.1 (0.101–0.109) 78.9–90.3 (0.082–0.230) 78.9–90.3 (0.082–0.230) 76.6–93.3 (0.104–0.297) 76.6–93.3 (0.104–0.297)
94.3–94.4 (0.021–0.023) 81.0–86.2 (0.162–0.206) 81.0–86.2 (0.162–0.206) 69.8–89.2 (0.119–0.202) 69.8–89.2 (0.119–0.202) 86.1 (0.172) 86.1 (0.172)
85.6–87.7 (0.047–0.049) 71.8–97.5 (0.014–0.235) 71.8–73.4 (0.213–0.235) 62.1–96.8 (0.043–4.136) 62.1–96.8 (0.043–4.136) 67.8–83.1 (0.161–0.184) 67.8–83.1 (0.161–0.184)
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9 6
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Fig. 3. Phylogenetic relationships between ticks found in and outside China. ML/Bayesian trees were based on the COI gene sequences. Numbers (>50%/>0.5) above or below branches indicate bootstrap values or posterior node probabilities. Argas persicus was used as an outgroup. The GenBank accession numbers of the sequences obtained in this study are shown in this figure.
was closely related to I. persulcatus (AB073725) from Japan and formed another lineage. Unlike in the 12S rRNA tree, I. scapularis (GU074891) from the USA had a closer relationship with I. ricinus ticks from northern Europe than with I. persulcatus from China. However, the support to this relationship was weak. Within the second phylogroup, there were 4 sub-phylogroups corresponding to the 4 genera: Amblyomma, Dermacentor, Haemaphysalis, and Rhipicephalus. However, a rather different picture of the evolutionary relationship among these 4 genera was observed in the phylogenies of the COI gene. Dermacentor ticks and Rhipicephalus ticks clustered together with the genus Amblyomma, and they formed a sister taxon of Haemaphysalis ticks. However, the support to this relationship was weak. Different from that in the 12S rRNA tree and the ITS2 tree (see below), notably, D. sinicus clustered together with D. marginatus from Romania (FN394329) in the COI tree. As D. sinicus is present in Hebei Province (Chen et al., 2010) which surrounds Beijing, further studies are needed to clarify the reason.
ITS2 gene The ITS2 sequences we obtained from the ticks collected in China varied in length from 882 bp in I. granulatus to 1803 bp in H. longicornis. The estimated mean frequencies of the 4 nucleotides were as follows: adenine 18.5–23.8%, cytosine 22.3–29.8%, thymidine 16.8–27.4%, and guanine 27.8–33.7%. The I. granulatus and I. persulcatus were 51% GC rich, whereas the rest of the ticks were 61–63% GC rich. The similarities of intraspecific sequences varied from 84.5% to 100%, while similarities of interspecific sequences within genera were estimated from 62.1% to 97.5% (Table 2). The similarities of intraspecific sequences of the ticks from different sampling locations were 84.5–100%, but the similarities of intraspecific sequences of the ticks from the same sampling location were 92.4–100%. In addition, samples of the 7 tick species we collected in China exhibited 0–15.5% differences from the same species or 2.5–37.9% differences from other species within the same genus from other parts of the world.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
7
Fig. 4. Phylogenetic relationships between ticks found in and outside China. The rooted tree was first inferred using the Bayesian MCMC method based on the ITS2 sequences. Accordingly, the basal lineage estimated by the MCC tree was used as the basal lineage for the phylogenetic trees constructed under the ML phylogenetic analyses. Numbers (>50%/>0.5) above or below branches indicate bootstrap values or posterior node probabilities. The GenBank accession numbers of the sequences obtained in this study are shown in this figure.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
8
As the ITS2 sequences of the soft tick (Argas species) were not available in GenBank, the rooted tree of the ITS2 sequences was first inferred using the Bayesian MCMC method (Drummond and Rambaut, 2007). In the ITS2 tree (Fig. 4), the clustering patterns of tick species were similar to those in the COI gene tree described above. All ticks of the genus Ixodes formed the first phylogroup, while other tick species formed the second phylogroup. Moreover, each of the 7 species formed a monospecific cluster. In addition, H. longicornis ticks from Gansu Province (FN296271 and HQ005301) were closely related to the ticks collected from Henan and Hubei provinces in this study. Similarly to the results reported by Tian et al. (2011), the species H. qinghaiensis from Gansu Province (HQ005302) was closely related to H. flava collected from Hubei Province.
Discussion Morphological examination identified the ticks we collected in endemic areas of tick-borne diseases in China to 7 species of 4 genera of the family Ixodidae. In agreement with the work by Chen et al. (2010), we found H. flava in Hubei, H. longicornis in Henan and Hubei, I. granulatus in Zhejiang, I. persulcatus in Inner Mongolia, and R. microplus in Henan and Hubei. Furthermore, our works indicated the presence of D. sinicus in Beijing and R. sanguineus in Zhejiang. Phylogenetic analyses of the 12S rRNA, COI, and ITS2 gene sequences verified the species identities of these ticks and further revealed the relationships of these ticks with the hard ticks in and outside China. Nava et al. (2010) found that the intraspecific genetic divergence among 16S rDNA sequences of A. dubitatum ticks collected in different localities from Argentina, Brazil, and Uruguay was much lower than the differences to the remaining Amblyomma species. Similarly, our results supported a high genetic conservation of ticks within the same species in the 12S rRNA gene sequence (Ketchum et al., 2009; Burlini et al., 2010), the COI gene sequence (Chitimia et al., 2010; Song et al., 2011), and the ITS2 gene sequence (Mtambo et al., 2007; Chao et al., 2011; Tian et al., 2011). The 7 species of ticks collected in China were highly homogeneous with the same species from other regions and fell into the corresponding lineage of recognized species (Figs. 2–4). Consistent with previous studies (Barker and Murrell, 2002, 2004), the phylogenetic analyses of 3 gene fragments (12S rRNA, COI, and ITS2) divided the 7 species of ticks into 2 phylogroups (Figs. 2–4). The first phylogroup contains Ixodes ticks, which can be further divided into 2 lineages, the Australian–New Guinea Ixodes and the other Ixodes (Klompen, 1999; Klompen et al., 2000; Barker and Murrell, 2002, 2004). In the present study, 2 Ixodes species (I. persulcatus and I. granulatus) were found in Inner Mongolia and Zhejiang province; both species fell into the second lineage. As expected, I. persulcatus from Inner Mongolia clustered together with the same species from Japan (Fig. 3) or Russia (Fig. 2). Interestingly, it shows a closer relationship with I. scapularis from North America than with I. ricinus from Europe in the 12S rRNA tree (Fig. 2). However, similarly to an earlier study based on the 16S rRNA gene (Caporale et al., 1995), a closer relationship between I. scapularis and I. ricinus was observed in both the COI and ITS2 trees (Figs. 3 and 4). As in an earlier study (Chao et al., 2011), I. granulatus from Zhejiang clustered together with I. granulatus from Japan and Taiwan of China, and formed a distinct sub-lineage in the present study. In the second phylogroup, the 5 species of ticks in China were compared with ticks (including Amblyomma ticks) from other parts of the world. Phylogenetic estimation based on COI or ITS2 gene sequences revealed 4 clades (Amblyomma, Dermacentor, Haemaphysalis, and Rhipicephalus) (Figs. 3 and 4). Furthermore, the
estimation produced the same topology in the COI tree or the ITS2 tree for the 4 genera. Similarly to former studies (Barker and Murrell, 2002, 2004), Dermacentor ticks were more closely related to Rhipicephalus ticks than to other genera. However, the topology in the 12S rRNA tree only displays 3 clades (Dermacentor, Amblyomma, Haemaphysalis, and Rhipicephalus). In the 12S rRNA tree, Amblyomma ticks clustered together with Haemaphysalis ticks. All Rhipicephalus ticks formed a genetically distinct clade (Fig. 2). In this genus, all R. microplus ticks including 7 from China clustered together. R. microplus from China were more closely related to R. microplus from Nepal (AF150042) and from India (EU921772) than with ticks found in other parts of the world (Australia and South America). The clustering patterns of these ticks were according to their geographical origins. In addition, all R. sanguineus ticks including 3 from Zhejiang clustered together. The ticks R. sanguineus from China were more closely related to R. sanguineus from Thailand (AY987377), followed by the tick from Brazil (EU346676), than with those found from other parts of the world (Europe and South America). However, the clustering patterns of these ticks were not according to their geographical origins. Several studies also concluded that the observed clustering patterns do not correspond well with the involved subjects’ geographic origins (Jorde and Wooding, 2004). In conclusion, the present study provides the first genetic characterization of the 12S rRNA, COI, and ITS2 genes of 7 species of hard tick collected in 5 provinces in China, where tick-borne diseases were reported recently. These 7 species of ticks were genetically closely related to the same species or to related species found in China and other parts of the world. Vector-borne and zoonotic pathogens have specific associations with their reservoir hosts. As important vectors of human and animal pathogens, ticks put people and animals at risk to known or unknown pathogens. Rapid and accurate identification and long-term monitoring of ticks will be significant for the prevention and control of tick-borne diseases in China. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grants 81290343 and 81273014), and by the State Key Laboratory for Infectious Disease Prevention and Control (Grants 2011SKLID101 and 2012SKLID309). References Andrews, R., Chilton, N., Beveridge, I., Spratt, D., Mayrhofer, G., 1992. Genetic markers for the identification of three Australian tick species at various stages in their life cycles. Int. J. Parasitol. 78, 366–368. Barker, S., Murrell, A., 2002. Phylogeny, evolution and historical zoogeography of ticks: a review of recent progress. Exp. Appl. Acarol. 28, 55–68. Barker, S., Murrell, A., 2004. Systematics and evolution of ticks with a list of valid genus and species names. Parasitology 129 (Suppl.), S15–S36. Beati, L., Keirans, J.E., 2001. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12s ribosomal DNA gene sequences and morphological characters. J. Parasitol. 87, 32–48. Bowman, A., Nuttall, P., 2008. Ticks: Biology, Disease and Control, first ed. Cambridge University Press, Cambridge. Burlini, L., Teixeira, K., Szabó, M., Famadas, K., 2010. Molecular dissimilarities of Rhipicephalus sanguineus (Acari: Ixodidae) in Brazil and its relation with samples throughout the world: is there a geographical pattern? Exp. Appl. Acarol. 50, 361–374. Caporale, D., Rich, S., Spielman, A., Telford, S.R., Kocher, T., 1995. Discriminating between Ixodes ticks by means of mitochondrial DNA sequences. Mol. Phylogenet. Evol. 4, 361–365. Chai, C., Lu, Q., Sun, J., Jiang, L., Ling, F., Zhang, L., Zheng, S., Zhang, H., Ge, J., 2010. Seroepidemiologic investigation on tick-borne diseases of humans and domestic animals in Zhejiang Province. Zhonghua Liu Xing Bing Xue Za Zhi. 31, 1144–1147. Chao, L.L., Wu, W.J., Shih, C.M., 2011. Species identification of Ixodes granulatus (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences. Exp. Appl. Acarol. 54, 51–63.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Chen, Z., Yang, X., Bu, F., Yang, X., Yang, X., Liu, J., 2010. Ticks (Acari: Ixodoidea: Argasidae, Ixodidae) of China. Exp. Appl. Acarol. 51, 393–404. Chitimia, L., Lin, R.Q., Cosoroaba, I., Braila, P., Song, H.Q., Zhu, X.Q., 2009. Molecular characterization of hard and soft ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA. Parasitol. Res. 105, 907–911. Chitimia, L., Lin, R.Q., Cosoroaba, I., Wu, X.Y., Song, H.Q., Yuan, Z.G., Zhu, X.Q., 2010. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes. Exp. Appl. Acarol. 52, 305–311. Drummond, A., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 8, 214. Fan, M., Wang, J., Jiang, Y., Zong, D., Lenz, B., Walker, D., 1987. Isolation of a spotted fever group Rickettsia from a patient and related ecologic investigations in Xinjiang Uygur Autonomous Region of China. J. Clin. Microbiol. 25, 628–632. ˜ A., Horak, Guglielmone, A., Robbins, R., Apanaskevich, D., Petney, T., Estrada-Pena, I., Shao, R., Barker, S., 2010. The Argasidae, Ixodidae and Nuttalliellidae (Acari: Ixodida) of the world: a list of valid species names. Magnolia Press, Auckland. Hao, Q., Hou, X., Geng, Z., Wan, K., 2011. Distribution of Borrelia burgdorferi sensu lato in China. J. Clin. Microbiol. 49, 647–650. Heyman, P., Cochez, C., Hofhuis, A., Sprong, H., Porter, S., Losson, B., Saegerman, C., Donoso-Mantke, O., Niedrig, M., Papa, A., 2010. A clear and present danger: tick-borne diseases in Europe. Expert. Rev. Anti. Infect. Ther. 8, 33–50. Huelsenbeck, J., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Jackson, J., Chilton, N.B., Beveridge, I., Morris, M., Andrews, R.H., 1998. An electrophoretic comparison of the Australian paralysis tick, Ixodes holocyclus Neumann, 1899, with I. cornuatus Roberts, 1960 (Acari: Ixodidae). Aust. J. Zool. 46, 109–117. Jackson, J., Chilton, N.B., Beveridge, I., Morris, M., Andrews, R.H., 2000. Genetic variation within the ticks Ixodes holocyclus and Ixodes cornuatus from south-eastern Australia. Int. J. Parasitol. 30, 1159–1166. Jiang, L., Mo, S., Zheng, S., Cheng, S., Xie, S., Meng, Z., Ye, X., 2006. Leptospira DNA of Lyme disease was first detected in tick in Zhejiang Province. Chin. J. Health Lab. Technol. 16, 30–31. Jinbo, U., Kato, T., Ito, M., 2011. Current progress in DNA barcoding and future implications for entomology. Entom. Sci. 14, 107–124. Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129, S3–S14. Jorde, L., Wooding, S., 2004. Genetic variation, classification and ‘race’. Nat. Genet. 36, S28–S33. Ketchum, H., Teel, P., Coates, C., Strey, O., Longnecker, M., 2009. Genetic variation in 12S and 16S mitochondrial rDNA genes of four geographically isolated populations of Gulf Coast ticks (Acari: Ixodidae). J. Med. Entomol. 46, 482–489. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Klompen, J., 1999. Phylogenetic relationships in the family Ixodidae with emphasis on the genus Ixodes (Parasitiformes: Ixodidae). In: Needham, G.R., Mitchell, R., Horn, D.J., Welbourn, W.C. (Eds.), Acarology IX Symposia. Ohio State University, Ohio, pp. 349–354. Klompen, J., Black IV, W.C., Keirans, J.E., Norris, D.E., 2000. Systematics and biogeography of hard ticks, a total evidence approach. Cladistics 16, 79–102. Leiby, D., 2011. Transfusion-transmitted Babesia spp.: bull’s-eye on Babesia microti. Clin. Microbiol. Rev. 24, 14–28. Lovisolo, O., Hull, R., Rösler, O., 2003. Coevolution of viruses with hosts and vectors and possible paleontology. Adv. Virus Res. 62, 325–379. Marrelli, M., Souza, L., Marques, R., Labruna, M., Matioli, S., Tonon, A., Ribolla, P., Marinotti, O., Schumaker, T., 2007. Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA. J. Med. Entomol. 44, 222–228. Mtambo, J., Madder, M., van Bortel, W., Berkvens, D., Backeljau, T., 2007. Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification. Exp. Appl. Acarol. 41, 115–128. Nava, S., Venzal, J., Labruna, M., Mastropaolo, M., González, E., Mangold, A., Guglielmone, A., 2010. Hosts, distribution and genetic divergence (16S rDNA) of Amblyomma dubitatum (Acari: Ixodidae). Exp. Appl. Acarol. 51, 335–351.
9
Parola, P., Paddock, C., Raoult, D., 2005. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin. Microbiol. Rev. 18, 719–756. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Pritt, B., Sloan, L., Johnson, D., Munderloh, U., Paskewitz, S., McElroy, K., McFadden, J., Binnicker, M., Neitzel, D., Liu, G., Nicholson, W., Nelson, C., Franson, J., Martin, S., Cunningham, S., Steward, C., Bogumill, K., Bjorgaard, M., Davis, J., McQuiston, J., Warshauer, D., Wilhelm, M., Patel, R., Trivedi, V., Eremeeva, M., 2011. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N. Engl. J. Med. 365, 422–429. Qi, C., Zhou, D., Liu, J., Cheng, Z., Zhang, L., Wang, L., Wang, Z., Yang, D., Wang, S., Chai, T., 2011. Detection of Babesia divergens using molecular methods in anemic patients in Shandong Province, China. Parasitol. Res. 109, 241–245. Shao, R., Barker, S., 2007. Mitochondrial genomes of parasitic arthropods: implications for studies of population genetics and evolution. Parasitology 134, 153–167. Shao, R., Campbell, N., Barker, S., 2001. Numerous gene rearrangements in the mitochondrial genome of the wallaby louse, Heterodoxus macropus (Phthiraptera). Mol. Biol. Evol. 18, 858–865. Shaw, M., Murrell, A., Barker, S., 2002. Low intraspecific variation in the rRNA internal transcribed spacer 2 (ITS2) of the Australian paralysis tick, Ixodes holocyclus. Parasitol. Res. 88, 247–252. Song, S., Shao, R., Atwell, R., Barker, S., Vankan, D., 2011. Phylogenetic and phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences. Int. J. Parasitol. 41, 871–880. Song, Z., Han, L., Zhao, H., Yan, D., Li, H., Zhang, L., Liu, S., Yue, L., 2004. Studies on epidemic disease of tick-borne encephalitis (TBE) surveillance and TBE virus isolated. Lett. Biotechnol. 15, 43–50. Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML Web servers. Syst. Biol. 75, 758–771. Sun, S., Aishan, M., Wang, X., Meng, W., Feng, C., Zhang, F., Hang, C., Hu, Z., Zhang, Y., 2009. Epidemiology and phylogenetic analysis of Crimean-Congo hemorrhagic fever viruses in Xinjiang, China. J. Clin. Microbiol. 47, 2536–2543. Swanson, S., Neitzel, D., Reed, K., Belongia, E., 2006. Coinfections acquired from Ixodes ticks. Clin. Microbiol. Rev. 19, 708–727. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tian, J., He, Y., Wei, P., Li, W., Jie, S.H., Yao, J.H., 2010. Retrospective analysis of clinical diagnosis of 21 cases of human ehrlichiosis. Chin. J. Infect. Dis. 28, 278–281. Tian, Z., Xie, J., Yin, H., Luo, J., Zhang, L., Zhang, P., Luo, J., 2011. Discrimination between Haemaphysalis longicornis and H. qinghaiensis based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2). Exp. Appl. Acarol. 54, 165–172. Uilenberg, G., Thiaucourt, F., Jongejan, F., 2004. On molecular taxonomy: what is in a name? Exp. Appl. Acarol. 32, 301–312. WHO, 2006. Lyme Borreliosis in Europe: Influences of Climate and Climate Change, Epidemiology, Ecology and Adaptation measures. World Health Organization, Regional Office for Europe, Copenhagen, Denmark. Yang, X., Chen, Z., Liu, J., 2008. The valid genus and species names of ticks (Acari: Ixodida: Argasidae, Ixodidae) in China. J. Hebei Norm. Univ. (Nat. Sci. Ed.) 32, 529–533. Zhang, L., Liu, Y., Ni, D., Li, Q., Yu, Y., Yu, X., Wan, K., Li, D., Liang, G., Jiang, X., Jing, H., Run, J., Luan, M., Fu, X., Zhang, J., Yang, W., Wang, Y., Dumler, J., Feng, Z., Ren, J., Xu, J., 2008. Nosocomial transmission of human granulocytic anaplasmosis in China. JAMA 300, 2263–2270. Zhang, Y.Z., Zhou, D.J., Xiong, Y., Chen, X.P., He, Y.W., Sun, Q., Yu, B., Li, J., Dai, Y.A., Tian, J.H., Qin, X.C., Jin, D., Cui, Z., Luo, X.L., Li, W., Lu, S., Wang, W., Peng, J.S., Guo, W.P., Li, M.H., Li, Z.J., Zhang, S., Chen, C., Wang, Y., de Jong, M.D., Xu, J., 2011. Hemorrhagic fever caused by a novel tick-borne Bunyavirus in Huaiyangshan, China. Zhonghua Liu Xing Bing Xue Za Zhi. 32, 209–220. Zhang, Y.Z., Zhou, D.J., Qin, X.C., Tian, J.H., Xiong, Y., Wang, J.B., Chen, X.P., Gao, D.Y., He, Y.W., Jin, D., Sun, Q., Guo, W.P., Wang, W., Yu, B., Li, J., Dai, Y.A., Li, W., Peng, J.S., Zhang, G.B., Zhang, S., Chen, X.M., Wang, Y., Li, M.H., Lu, X., Ye, C., de Jong, M.D., Xu, J., 2012. The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China. J. Virol. 86, 2864–2868.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
ARTICLE IN PRESS Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Molecular survey of hard ticks in endemic areas of tick-borne diseases in China Xin Lu a,1 , Xian-Dan Lin b,1 , Jian-Bo Wang a,1 , Xin-Cheng Qin a , Jun-Hua Tian c , Wen-Ping Guo a , Fei-Neng Fan d , Renfu Shao e , Jianguo Xu a , Yong-Zhen Zhang a,∗ a State Key Laboratory for Infectious Disease Prevention and Control, Department of Zoonoses, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping Liuzi 5, 102206 Beijing, China b Wenzhou Center for Disease Control and Prevention, 325001 Wenzhou, Zhejiang Province, China c Wuhan Center for Disease Control and Prevention, 430022 Wuhan, Hubei Province, China d Cixi Center for Disease Control and Prevention, Cixi 315300, Zhejiang Province, China e GeneCology Research Group, School of Science, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland, Australia
a r t i c l e
i n f o
Article history: Received 25 July 2012 Received in revised form 22 December 2012 Accepted 9 January 2013 Available online xxx Keywords: Ticks Taxonomy Molecular analysis Phylogeny China
a b s t r a c t Over the past several years, there was a substantial increase in the number of cases of known and novel tick-borne infections in humans in China. To better understand the ticks associated with these infections, we collected hard ticks from animals or around livestock shelters in 29 localities in 5 provinces (Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang) where cases of tick-borne illness were reported. We collected 2950 hard ticks representing 7 species of 4 genera (Dermacentor sinicus, Haemaphysalis flava, Haemaphysalis longicornis, Ixodes granulatus, Ixodes persulcatus, Rhipicephalus microplus, and Rhipicephalus sanguineus). These ticks were identified to species using morphological characters initially. We then sequenced the mitochondrial small subunit rRNA (12S rRNA) gene, cytochrome oxidase subunit 1 (COI) gene, and the second internal transcribed spacer (ITS2) gene of these ticks, and conducted phylogenetic analyses. Our analyses showed that the molecular and morphological data are consistent in the identification of the 7 tick species. Furthermore, all these 7 tick species from China were genetically closely related to the same species or related species found outside China. Rapid and accurate identification and long-term monitoring of these ticks will be of significance to the prevention and control of tick-borne diseases in China. © 2013 Published by Elsevier GmbH.
Introduction Ticks (Acari: Ixodida) are parasitic arachnids with a worldwide distribution. The Ixodida have 3 families, Argasidae (soft ticks), Nuttalliellidae, and Ixodidae (hard ticks), with 907 described species (Bowman and Nuttall, 2008; Guglielmone et al., 2010). Ticks act as reservoirs and vectors of a broad range of pathogens for humans and animals, including viruses, bacteria, rickettsiae, and protozoans (Parola et al., 2005; Swanson et al., 2006; Heyman et al., 2010; Leiby, 2011). These tick-borne pathogens are responsible for more than 100,000 cases of illness in humans each year throughout the world (WHO, 2006). Approximately 10% of the currently recognized tick species are known to carry pathogens of humans and animals (Jongejan and Uilenberg, 2004). Some previously unrecognized or emerging pathogens have been identified recently in ticks, such as Huaiyangshan virus (Zhang et al., 2011) and Ehrlichia species (Pritt et al., 2011). Moreover, zoonotic or vector-borne pathogens
∗ Corresponding author. Tel.: +86 10 58900782; fax: +86 10 58900700. E-mail address: [email protected] (Y.-Z. Zhang). 1 These authors contributed equally to this work and share first authorship.
(especially viruses) show specific association with their hosts (Lovisolo et al., 2003). Traditionally, species identification of ticks has been mainly based on morphological examination. As special skills acquired through extensive experience are needed, only a limited number of experts such as taxonomists and trained technicians can identify ticks to species accurately (Jinbo et al., 2011). Genetic and phylogenetic analyses based on DNA sequences could reveal species groups and assign unknown individuals to species (Uilenberg et al., 2004), and infer the evolutionary relationship of ticks within and between species. Hence, genetic and phylogenetic analyses of molecular markers have been extensively used to identify species and understand phylogenetic relationships of ticks in the past 2 decades (Andrews et al., 1992; Jackson et al., 1998, 2000; Shaw et al., 2002; Song et al., 2011). Several genes, such as the mitochondrial 12S ribosomal RNA gene (12S rRNA), the cytochrome oxidase subunit 1 (CO1), and the second internal transcribed spacer (ITS2), have been used frequently for taxonomical and evolutionary investigation (Barker and Murrell, 2002, 2004; Shao and Barker, 2007; Jinbo et al., 2011). Moreover, DNA (and RNA) extracted from ticks carrying tick-borne diseases collected in endemic areas could be analyzed to identify both etiologic agents and vector tick species as
1877-959X/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
2
well (Marrelli et al., 2007; Zhang et al., 2012). Molecular analyses, thus, are very useful and practical tools for monitoring ticks that are of public health significance. At least 117 species of ticks (by morphological examination) are known to occur in China; these ticks are in 10 genera of 2 families and have a nationwide distribution (Yang et al., 2008; Chen et al., 2010). Cases of well-known tick-borne diseases, such as Lyme disease (Hao et al., 2011), spotted fever (Fan et al., 1987), CrimeanCongo hemorrhagic fever (Sun et al., 2009), and babesiosis (Qi et al., 2011), have been reported from China. In the past several years, there was a substantial increase in the number of the cases of known and novel tick-borne infections in humans in China. Furthermore, the illness occurred in a large area from the north of Inner Mongolia to the south of Hubei province. Recently, human granulocytic anaplasmosis transmitted by ticks was reported from Anhui Province of China (Zhang et al., 2008). Notably, a new hemorrhagic fever-like disease with up to 15% mortality caused by Huaiyangshan virus (HYSV) (a novel tick-borne Bunyavirus) occurred in Henan and Hubei provinces of China in 2009 (Zhang et al., 2011). Further investigation indicated that both Haemaphysalis longicornis and Rhipicephalus microplus can carry HYSV, and the abundance of H. longicornis appears to determine the emergence of the new disease (Zhang et al., 2012). As very few molecular studies on ticks have been conducted in China, most of the 117 species of ticks described from China were identified by morphological examination (Yang et al., 2008; Chen et al., 2010). Thus, far less is known about the phylogenetic relationships between ticks in and outside China. For controlling emerging or reemerging tick-borne diseases and minimizing the impact of them, it is urgent to better understand the molecular data and the phylogenetic relationships of the ticks collected in endemic areas of tick-borne diseases in China. In this study, we identified 7 species of ticks collected from endemic areas of tick-borne diseases in China and conducted molecular phylogenetic analyses among these ticks and other ticks previously identified in China and other parts of the world. Materials and methods Ticks collection In 2010, 2950 adult ticks (males: 882; females: 2068) were collected from animals or around livestock shelters at 29 localities of 5 provinces in China: Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang (Fig. 1). They were collected from hedgehogs (Erinaceus amurensis), sheep (Ovis aries), cattle (Bos taurus), goats (Capra aegagrus hircus), and dogs (Canis lupus familiaris). All ticks were first identified morphologically under light microscope by a trained technician and then verified by molecular analysis.
PCR reactions were performed in 50-l volumes including 2 l DNA (100 ng) and 2.5 U of rTaq DNA polymerase (Takara, Dalian, China). The cycling conditions were: 94 ◦ C for 5 min (initial denaturation) followed by 35 cycles of 94 ◦ C for 30 s (denaturation), 55 ◦ C for 1 min (annealing), 72 ◦ C for 1 min (extension), and a final extension of 72 ◦ C for 10 min. A negative control (no DNA template) was included in each amplification run. Successful amplification was determined by electrophoresing 2 l of the PCR amplicons on 1.5% (w/v) agarose gels (1 × TBE) and visualizing with gold view under UV light. The DL2000 DNA size marker (Takara) was included in the agarose gels. There was only one single band of expected size (approximately 380 bp for the 12S rRNA gene, 660 bp for the COI gene, and 920–1850 bp for the ITS2 gene) in each PCR amplification. PCR amplicons sequencing PCR amplicons were purified using the QIAquick Gel Extraction kit (Qiagen) according to the manufacturer’s recommendations. The purified DNA (<700 bp) was subjected to a direct sequencing. The sequencing primers were the same as the PCR amplification primers. But the purified DNA (>700 bp) was cloned into pMD18-T vector (TaKaRa). Then, the vector was transformed into JM109competent cells. At least 3 DNA clones were used for sequencing by M13 sequencing primers. DNA sequencing was performed using Applied Biosystems 377 gene sequencers by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Automatic DNA sequence assembly was performed by DNA baser software, version 3.2.5. Both the forward and reverse reads were used for sequence assembly; primer sequences were then excluded with SeqMan (DNAstar Inc.). Sequence alignment CLUSTAL X2 software (version 2.0.12) was used to align all sequences obtained in this study and retrieved from GenBank with the multiple alignment modes. For comparing genetic and phylogenetic analysis, all sequences (primer sequences excluded) were kept in the same size after editing with MEGA5. The following dataset sizes were used in the final phylogenetic analysis: 12S rRNA = 72 sequences, 330 nt; COI = 40 sequences, 546 nt; ITS2 = 58 sequences, 852 nt. The nucleotide similarities were calculated using MegAlign (DNAstar Inc.). Calculating maximum likelihood estimate of transition/transversion bias was performed under the Tamura–Nei model with MEGA5 (Tamura et al., 2011). The value of pairwise distance between sequences was calculated in MEGA5 using the Kimura 2parameter model (Kimura, 1980). Standard error estimates were obtained by a bootstrap procedure (1000 replicates). All ambiguous positions were removed for each sequence pair.
PCR amplification of 12S rRNA, COI and ITS2 genes
Phylogenetic analysis
After washing twice with phosphate-buffered saline, ticks were homogenized with a mortar and pestle in 1 ml of phosphatebuffered saline. A total of 200 l of homogenates was used to extract DNA with the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. All DNA samples were diluted to 50 ng/l and then subjected to PCR for amplification. Fragments of the 12S rRNA gene, the COI gene and the ITS2 gene were amplified from each tick DNA sample with primer pairs T1B (5 -AAACTAGGATTAGATACCCT-3 ) and T2A (5 -AATGAGAGCGACGGGCGATGT-3 ) (Beati and Keirans, 2001), C1-J-1718 (5 -GGAGGATTTGGAAATTGATTAGTTCC-3 ) and C1-N2329 (5 -ACTGTAAATATATGATGAGCTCA-3 ) (Shao et al., 2001) and TITS2F1 (5 -CGAGACTTGGTGTGAATTGCA-3 ) and TITS2R1 (5 TCCCATACACCACATTTCCCG-3 ) (Chitimia et al., 2009), respectively.
Phylogenetic relationships were estimated with the Bayesian method and the maximum likelihood (ML) method with 1000 replications in the bootstrap test. The Bayesian trees were constructed with Mrbayes v3.1.2 (Huelsenbeck and Ronquist, 2001), and the ML analyses were performed through (Stamatakis http://phylobench.vital-it.ch/raxml-bb/index.php et al., 2008). The model used to construct the trees was chosen by the Modeltest program (version 3.7) (Posada and Crandall, 1998). When trees were constructed based on the 12S rRNA and COI gene sequences, soft tick (Argas species) sequences were used as an outgoup. As the ITS2 sequence of the soft tick was not available in GenBank, the rooted tree of the ITS2 sequences was inferred using the Bayesian MCMC method available in the BEAST v1.6.0 package (Drummond and Rambaut, 2007). Because the MCC tree
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
3
Fig. 1. Locations where ticks were collected in this study. Ticks were collected in Beijing, Henan, Hubei, Inner Mongolia, and Zhejiang provinces. The locations are indicated by triangles.
is automatically rooted on the assumption of a molecular clock, it enables determination of the basal lineage. Accordingly, the basal lineage estimated by the MCC tree was used as the basal lineage to root the phylogenetic trees inferred under the ML phylogenetic analyses. Results Ticks samples In this study, a total of 2950 adult ticks (male: 882; female: 2068) was collected from 5 provinces in China (Table 1, Fig. 1), from where tick-borne diseases (tick-borne encephalitis, Huaiyangshan hemorrhagic fever, Lyme disease, human granulocytic anaplasmosis, and other tick-borne rickettsioses) have been reported (Song et al., 2004; Jiang et al., 2006; Chai et al., 2010; Tian et al., 2010; Zhang et al., 2011). Morphological examination identified these ticks to 7 species of 4 genera of the family Ixodidae: Dermacentor sinicus, Haemaphysalis flava, Haemaphysalis longicornis, Ixodes granulatus, Ixodes persulcatus, Rhipicephalus microplus, and Rhipicephalus sanguineus, respectively. The species, their geographical origins, and GenBank accession numbers of these tick sequences are shown in Figs. 2–4. 12S rRNA gene For all 12S rRNA sequences obtained in China, the sequences varied in length from 337 bp in H. flava to 349 bp in I. persulcatus (excluding the primers). The estimated mean frequencies of the 4 nucleotides were variable for the 7 species as follows: adenine 38.9–43.9%, cytosine 6.6–8.7%, thymidine 33.7–40.5%, and guanine 12–14.8%. The similarities of intraspecific sequences varied from
81% to 100%, while similarities of interspecific sequences within a given genus were estimated from 76.6% to 96.8% (Table 2). The differences of intraspecific sequences among the ticks from different sampling locations were 0–19%, but the differences of intraspecific sequences from the same sampling location were 0–1.9%. In addition, the tick species identified in the present study in China exhibited 0–14.8% differences from the same species or 3.2–23.4% from other species in the same genus from other parts of the world. To investigate the evolutionary relationships between ticks collected in China and around the world, we constructed phylogenetic trees using the 12S rRNA gene sequences obtained in this study and reported previously (retrieved from GenBank) with both Bayesian and ML methods (Fig. 2). A soft tick, Argas reflexus, was used as the outgroup. Consistent with the traditional taxonomy, the ticks collected in this study were grouped into 4 genera, Ixodes, Dermacentor, Haemaphysalis, and Rhipicephalus, with strong support, 0.96, 1.0, 1.0, and 1.0, respectively. All Ixodes ticks were clustered together and formed a phylogroup in both the Bayesian and ML trees, while the ticks from the other 3 genera were grouped into another phylogroup. Within the first phylogroup, as expected, I. persulcatus collected from Inner Mongolia of China was closely related to I. persulcatus (HM234631) from Russia. Furthermore, the species had a closer relationship with I. scapularis (L43901) from the USA, followed by I. ricinus ticks which are distributed widely in Europe. Interestingly, the 3 samples of I. granulatus from Zhejiang Province formed a lineage, and had a closer relationship with I. persulcatus, I. scapularis, and I. ricinus than with I. uriae from Iceland (AM410581). Within the second phylogroup, H. flava and H. longicornis collected from Henan and Hubei provinces of China, where a novel hemorrhagic fever disease transmitted by H. longicornis had been reported in 2009 (Zhang et al., 2011), were closely related to each
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9 4
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Fig. 2. Phylogenetic relationships between ticks found in and outside China. ML/Bayesian trees were based on the 12S rRNA gene sequences. Numbers (>50%/>0.5) above or below branches indicate bootstrap values or posterior node probabilities. Argas reflexus was used as an outgroup. The GenBank accession numbers of the sequences obtained in this study are shown in this figure.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
5
Table 1 The species and geographical locations of tick specimens collected from animal hosts and livestock shelters in this study. Species
Hosts
No. of ticks (male/female) Location
D. sinicus H. flava H. longicornis
I. granulatus I. persulcatus R. microplus
R. sanguineus
Hedgehogs Cattle Dogs Cattle Goats Dogs Livestock shelter Sheep Livestock shelter Cattle Goats Dogs Cattle Goats Livestock shelter
Total
Total
Beijing
Henan
Hubei
Inner Mongolia
Zhejiang
19/37 – – – – – – – – – – – – – –
– – – 79/148 8/18 5/14 – – – 164/329 – – – – –
– 31/58 9/15 42/178 13/42 6/12 – – – 397/936 7/20 2/9 – – –
– – – – – – – 31/72 23/55 – – – – – –
– – – – – – 4/11 – – – – – 25/62 15/41 2/11
19/37 31/58 9/15 121/326 21/60 11/26 4/11 31/72 23/55 561/1265 7/20 2/9 25/62 15/41 2/11
19/37
256/509
507/1270
54/127
46/125
882/2068
other and formed a lineage. These 2 species had a closer relationship (and formed a sub-phylogroup) with Amblyomma species from America and Africa than with ticks of the genera Dermacentor and Rhipicephalus. In the genus Dermacentor, the 4 samples of D. sinicus collected in Beijing clustered together. D. sinicus from China was closely related to D. marginatus from Italy and Switzerland. D. sinicus and D. marginatus had a closer relationship with D. reticulatus (AF150038) from France, and formed a sister taxon of D. andersoni (AF150040) and D. variabilis (AF031851). All Rhipicephalus ticks, including samples of Rhipicephalus species from China and those from other parts of the world, were clustered together and could be divided into 2 lineages. Within the first lineage, R. sanguineus from China was more closely related to R. sanguineus from Thailand (AY987377) and Brazil (EU346676) than those from other parts of the world. Furthermore, R. sanguineus had a close relationship with R. appendiculatus (AF150028) from Uganda. R. microplus ticks collected from Henan and Hubei provinces of China were clustered together and were closely related to R. microplus from other Asian countries, South America, Africa, and Australia. R. microplus had a close relationship with R. kohlsi from Jordan (AF150043), and they formed another lineage with R. pravus and R. kochi from Tanzania.
COI gene The COI sequences obtained from the ticks in China were approximately 610 bp (excluding the primers) in length. The estimated mean frequencies of the 4 nucleotides were as follows: adenine 27.6–31.4%, cytosine 17–19.1%, thymidine 37.4–40.6%, and guanine 13.7–14.9%. The similarities of intraspecific sequences varied from 93.9% to 100%, while similarities of interspecific sequences within genera were estimated from 69.8% to 94.4% (Table 2). The similarities of intraspecific sequences of the ticks from different sampling locations were 93.9–100%, whereas the similarities of intraspecific sequences from the same sampling location were 97.7–100%. In addition, the tick species identified in this study were closely related to the same species (0–6.1% differences), but exhibited 5.6–30.2% differences from other species in the same genus. The phylogenetic relationships among these ticks was inferred with COI gene sequences using the soft tick (A. persicus) as the outgroup (Fig. 3). Consistent with the 12S rRNA trees, the ticks were also divided into 2 phylogroups in the COI trees. The first phylogroup contained only the ticks of the genus Ixodes, while the other 4 genera of ticks formed the second phylogroup. Within the first phylogroup, I. granulatus from Zhejiang Province formed a distinct lineage. I. persulcatus collected from Inner Mongolia of China
Table 2 The average value of pairwise distance and the range of the similarities between sequences of ticks from the same or from different species. Species
Similarities between sequences/(genetic distance between sequences) Within species
D. sinicus H. flava H. longicornis I. granulatus I. persulcatus R. microplus R. sanguineus
Between species
12S rDNA
COI
ITS2
12S rDNA
COI
ITS2
99.5–100.0 0 100 (0–0.003) 99.5–100.0 (0–0.003) 98.6–99.1 (0.004–0.007) 97.9–100.0 0 97.7–100.0 (0–0.028) 81–100.0 (0–0.087)
99.8–100.0 (0–0.002) 99.8–100.0 (0–0.002) 93.9–100.0 (0–0.027) 99.5–99.7 (0.006) 99.7–100.0 (0–0.004) 99.7–100.0 (0–0.003) 100 0
97.7–99.4 0 98.1–100.0 0 89.1–99.4 (0.001–0.017) 89.7–100.0 (0–0.005) 87–97.6 (0–0.009) 89–99.7 (0–0.017) 84.5–100.0 (0–0.009)
83.5–96.8 (0.041–0.166) 91.6–92.1 (0.101–0.109) 91.6–92.1 (0.101–0.109) 78.9–90.3 (0.082–0.230) 78.9–90.3 (0.082–0.230) 76.6–93.3 (0.104–0.297) 76.6–93.3 (0.104–0.297)
94.3–94.4 (0.021–0.023) 81.0–86.2 (0.162–0.206) 81.0–86.2 (0.162–0.206) 69.8–89.2 (0.119–0.202) 69.8–89.2 (0.119–0.202) 86.1 (0.172) 86.1 (0.172)
85.6–87.7 (0.047–0.049) 71.8–97.5 (0.014–0.235) 71.8–73.4 (0.213–0.235) 62.1–96.8 (0.043–4.136) 62.1–96.8 (0.043–4.136) 67.8–83.1 (0.161–0.184) 67.8–83.1 (0.161–0.184)
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9 6
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Fig. 3. Phylogenetic relationships between ticks found in and outside China. ML/Bayesian trees were based on the COI gene sequences. Numbers (>50%/>0.5) above or below branches indicate bootstrap values or posterior node probabilities. Argas persicus was used as an outgroup. The GenBank accession numbers of the sequences obtained in this study are shown in this figure.
was closely related to I. persulcatus (AB073725) from Japan and formed another lineage. Unlike in the 12S rRNA tree, I. scapularis (GU074891) from the USA had a closer relationship with I. ricinus ticks from northern Europe than with I. persulcatus from China. However, the support to this relationship was weak. Within the second phylogroup, there were 4 sub-phylogroups corresponding to the 4 genera: Amblyomma, Dermacentor, Haemaphysalis, and Rhipicephalus. However, a rather different picture of the evolutionary relationship among these 4 genera was observed in the phylogenies of the COI gene. Dermacentor ticks and Rhipicephalus ticks clustered together with the genus Amblyomma, and they formed a sister taxon of Haemaphysalis ticks. However, the support to this relationship was weak. Different from that in the 12S rRNA tree and the ITS2 tree (see below), notably, D. sinicus clustered together with D. marginatus from Romania (FN394329) in the COI tree. As D. sinicus is present in Hebei Province (Chen et al., 2010) which surrounds Beijing, further studies are needed to clarify the reason.
ITS2 gene The ITS2 sequences we obtained from the ticks collected in China varied in length from 882 bp in I. granulatus to 1803 bp in H. longicornis. The estimated mean frequencies of the 4 nucleotides were as follows: adenine 18.5–23.8%, cytosine 22.3–29.8%, thymidine 16.8–27.4%, and guanine 27.8–33.7%. The I. granulatus and I. persulcatus were 51% GC rich, whereas the rest of the ticks were 61–63% GC rich. The similarities of intraspecific sequences varied from 84.5% to 100%, while similarities of interspecific sequences within genera were estimated from 62.1% to 97.5% (Table 2). The similarities of intraspecific sequences of the ticks from different sampling locations were 84.5–100%, but the similarities of intraspecific sequences of the ticks from the same sampling location were 92.4–100%. In addition, samples of the 7 tick species we collected in China exhibited 0–15.5% differences from the same species or 2.5–37.9% differences from other species within the same genus from other parts of the world.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
7
Fig. 4. Phylogenetic relationships between ticks found in and outside China. The rooted tree was first inferred using the Bayesian MCMC method based on the ITS2 sequences. Accordingly, the basal lineage estimated by the MCC tree was used as the basal lineage for the phylogenetic trees constructed under the ML phylogenetic analyses. Numbers (>50%/>0.5) above or below branches indicate bootstrap values or posterior node probabilities. The GenBank accession numbers of the sequences obtained in this study are shown in this figure.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
8
As the ITS2 sequences of the soft tick (Argas species) were not available in GenBank, the rooted tree of the ITS2 sequences was first inferred using the Bayesian MCMC method (Drummond and Rambaut, 2007). In the ITS2 tree (Fig. 4), the clustering patterns of tick species were similar to those in the COI gene tree described above. All ticks of the genus Ixodes formed the first phylogroup, while other tick species formed the second phylogroup. Moreover, each of the 7 species formed a monospecific cluster. In addition, H. longicornis ticks from Gansu Province (FN296271 and HQ005301) were closely related to the ticks collected from Henan and Hubei provinces in this study. Similarly to the results reported by Tian et al. (2011), the species H. qinghaiensis from Gansu Province (HQ005302) was closely related to H. flava collected from Hubei Province.
Discussion Morphological examination identified the ticks we collected in endemic areas of tick-borne diseases in China to 7 species of 4 genera of the family Ixodidae. In agreement with the work by Chen et al. (2010), we found H. flava in Hubei, H. longicornis in Henan and Hubei, I. granulatus in Zhejiang, I. persulcatus in Inner Mongolia, and R. microplus in Henan and Hubei. Furthermore, our works indicated the presence of D. sinicus in Beijing and R. sanguineus in Zhejiang. Phylogenetic analyses of the 12S rRNA, COI, and ITS2 gene sequences verified the species identities of these ticks and further revealed the relationships of these ticks with the hard ticks in and outside China. Nava et al. (2010) found that the intraspecific genetic divergence among 16S rDNA sequences of A. dubitatum ticks collected in different localities from Argentina, Brazil, and Uruguay was much lower than the differences to the remaining Amblyomma species. Similarly, our results supported a high genetic conservation of ticks within the same species in the 12S rRNA gene sequence (Ketchum et al., 2009; Burlini et al., 2010), the COI gene sequence (Chitimia et al., 2010; Song et al., 2011), and the ITS2 gene sequence (Mtambo et al., 2007; Chao et al., 2011; Tian et al., 2011). The 7 species of ticks collected in China were highly homogeneous with the same species from other regions and fell into the corresponding lineage of recognized species (Figs. 2–4). Consistent with previous studies (Barker and Murrell, 2002, 2004), the phylogenetic analyses of 3 gene fragments (12S rRNA, COI, and ITS2) divided the 7 species of ticks into 2 phylogroups (Figs. 2–4). The first phylogroup contains Ixodes ticks, which can be further divided into 2 lineages, the Australian–New Guinea Ixodes and the other Ixodes (Klompen, 1999; Klompen et al., 2000; Barker and Murrell, 2002, 2004). In the present study, 2 Ixodes species (I. persulcatus and I. granulatus) were found in Inner Mongolia and Zhejiang province; both species fell into the second lineage. As expected, I. persulcatus from Inner Mongolia clustered together with the same species from Japan (Fig. 3) or Russia (Fig. 2). Interestingly, it shows a closer relationship with I. scapularis from North America than with I. ricinus from Europe in the 12S rRNA tree (Fig. 2). However, similarly to an earlier study based on the 16S rRNA gene (Caporale et al., 1995), a closer relationship between I. scapularis and I. ricinus was observed in both the COI and ITS2 trees (Figs. 3 and 4). As in an earlier study (Chao et al., 2011), I. granulatus from Zhejiang clustered together with I. granulatus from Japan and Taiwan of China, and formed a distinct sub-lineage in the present study. In the second phylogroup, the 5 species of ticks in China were compared with ticks (including Amblyomma ticks) from other parts of the world. Phylogenetic estimation based on COI or ITS2 gene sequences revealed 4 clades (Amblyomma, Dermacentor, Haemaphysalis, and Rhipicephalus) (Figs. 3 and 4). Furthermore, the
estimation produced the same topology in the COI tree or the ITS2 tree for the 4 genera. Similarly to former studies (Barker and Murrell, 2002, 2004), Dermacentor ticks were more closely related to Rhipicephalus ticks than to other genera. However, the topology in the 12S rRNA tree only displays 3 clades (Dermacentor, Amblyomma, Haemaphysalis, and Rhipicephalus). In the 12S rRNA tree, Amblyomma ticks clustered together with Haemaphysalis ticks. All Rhipicephalus ticks formed a genetically distinct clade (Fig. 2). In this genus, all R. microplus ticks including 7 from China clustered together. R. microplus from China were more closely related to R. microplus from Nepal (AF150042) and from India (EU921772) than with ticks found in other parts of the world (Australia and South America). The clustering patterns of these ticks were according to their geographical origins. In addition, all R. sanguineus ticks including 3 from Zhejiang clustered together. The ticks R. sanguineus from China were more closely related to R. sanguineus from Thailand (AY987377), followed by the tick from Brazil (EU346676), than with those found from other parts of the world (Europe and South America). However, the clustering patterns of these ticks were not according to their geographical origins. Several studies also concluded that the observed clustering patterns do not correspond well with the involved subjects’ geographic origins (Jorde and Wooding, 2004). In conclusion, the present study provides the first genetic characterization of the 12S rRNA, COI, and ITS2 genes of 7 species of hard tick collected in 5 provinces in China, where tick-borne diseases were reported recently. These 7 species of ticks were genetically closely related to the same species or to related species found in China and other parts of the world. Vector-borne and zoonotic pathogens have specific associations with their reservoir hosts. As important vectors of human and animal pathogens, ticks put people and animals at risk to known or unknown pathogens. Rapid and accurate identification and long-term monitoring of ticks will be significant for the prevention and control of tick-borne diseases in China. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grants 81290343 and 81273014), and by the State Key Laboratory for Infectious Disease Prevention and Control (Grants 2011SKLID101 and 2012SKLID309). References Andrews, R., Chilton, N., Beveridge, I., Spratt, D., Mayrhofer, G., 1992. Genetic markers for the identification of three Australian tick species at various stages in their life cycles. Int. J. Parasitol. 78, 366–368. Barker, S., Murrell, A., 2002. Phylogeny, evolution and historical zoogeography of ticks: a review of recent progress. Exp. Appl. Acarol. 28, 55–68. Barker, S., Murrell, A., 2004. Systematics and evolution of ticks with a list of valid genus and species names. Parasitology 129 (Suppl.), S15–S36. Beati, L., Keirans, J.E., 2001. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12s ribosomal DNA gene sequences and morphological characters. J. Parasitol. 87, 32–48. Bowman, A., Nuttall, P., 2008. Ticks: Biology, Disease and Control, first ed. Cambridge University Press, Cambridge. Burlini, L., Teixeira, K., Szabó, M., Famadas, K., 2010. Molecular dissimilarities of Rhipicephalus sanguineus (Acari: Ixodidae) in Brazil and its relation with samples throughout the world: is there a geographical pattern? Exp. Appl. Acarol. 50, 361–374. Caporale, D., Rich, S., Spielman, A., Telford, S.R., Kocher, T., 1995. Discriminating between Ixodes ticks by means of mitochondrial DNA sequences. Mol. Phylogenet. Evol. 4, 361–365. Chai, C., Lu, Q., Sun, J., Jiang, L., Ling, F., Zhang, L., Zheng, S., Zhang, H., Ge, J., 2010. Seroepidemiologic investigation on tick-borne diseases of humans and domestic animals in Zhejiang Province. Zhonghua Liu Xing Bing Xue Za Zhi. 31, 1144–1147. Chao, L.L., Wu, W.J., Shih, C.M., 2011. Species identification of Ixodes granulatus (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences. Exp. Appl. Acarol. 54, 51–63.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003
G Model TTBDIS-201; No. of Pages 9
ARTICLE IN PRESS X. Lu et al. / Ticks and Tick-borne Diseases xxx (2013) xxx–xxx
Chen, Z., Yang, X., Bu, F., Yang, X., Yang, X., Liu, J., 2010. Ticks (Acari: Ixodoidea: Argasidae, Ixodidae) of China. Exp. Appl. Acarol. 51, 393–404. Chitimia, L., Lin, R.Q., Cosoroaba, I., Braila, P., Song, H.Q., Zhu, X.Q., 2009. Molecular characterization of hard and soft ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA. Parasitol. Res. 105, 907–911. Chitimia, L., Lin, R.Q., Cosoroaba, I., Wu, X.Y., Song, H.Q., Yuan, Z.G., Zhu, X.Q., 2010. Genetic characterization of ticks from southwestern Romania by sequences of mitochondrial cox1 and nad5 genes. Exp. Appl. Acarol. 52, 305–311. Drummond, A., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 8, 214. Fan, M., Wang, J., Jiang, Y., Zong, D., Lenz, B., Walker, D., 1987. Isolation of a spotted fever group Rickettsia from a patient and related ecologic investigations in Xinjiang Uygur Autonomous Region of China. J. Clin. Microbiol. 25, 628–632. ˜ A., Horak, Guglielmone, A., Robbins, R., Apanaskevich, D., Petney, T., Estrada-Pena, I., Shao, R., Barker, S., 2010. The Argasidae, Ixodidae and Nuttalliellidae (Acari: Ixodida) of the world: a list of valid species names. Magnolia Press, Auckland. Hao, Q., Hou, X., Geng, Z., Wan, K., 2011. Distribution of Borrelia burgdorferi sensu lato in China. J. Clin. Microbiol. 49, 647–650. Heyman, P., Cochez, C., Hofhuis, A., Sprong, H., Porter, S., Losson, B., Saegerman, C., Donoso-Mantke, O., Niedrig, M., Papa, A., 2010. A clear and present danger: tick-borne diseases in Europe. Expert. Rev. Anti. Infect. Ther. 8, 33–50. Huelsenbeck, J., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Jackson, J., Chilton, N.B., Beveridge, I., Morris, M., Andrews, R.H., 1998. An electrophoretic comparison of the Australian paralysis tick, Ixodes holocyclus Neumann, 1899, with I. cornuatus Roberts, 1960 (Acari: Ixodidae). Aust. J. Zool. 46, 109–117. Jackson, J., Chilton, N.B., Beveridge, I., Morris, M., Andrews, R.H., 2000. Genetic variation within the ticks Ixodes holocyclus and Ixodes cornuatus from south-eastern Australia. Int. J. Parasitol. 30, 1159–1166. Jiang, L., Mo, S., Zheng, S., Cheng, S., Xie, S., Meng, Z., Ye, X., 2006. Leptospira DNA of Lyme disease was first detected in tick in Zhejiang Province. Chin. J. Health Lab. Technol. 16, 30–31. Jinbo, U., Kato, T., Ito, M., 2011. Current progress in DNA barcoding and future implications for entomology. Entom. Sci. 14, 107–124. Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129, S3–S14. Jorde, L., Wooding, S., 2004. Genetic variation, classification and ‘race’. Nat. Genet. 36, S28–S33. Ketchum, H., Teel, P., Coates, C., Strey, O., Longnecker, M., 2009. Genetic variation in 12S and 16S mitochondrial rDNA genes of four geographically isolated populations of Gulf Coast ticks (Acari: Ixodidae). J. Med. Entomol. 46, 482–489. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Klompen, J., 1999. Phylogenetic relationships in the family Ixodidae with emphasis on the genus Ixodes (Parasitiformes: Ixodidae). In: Needham, G.R., Mitchell, R., Horn, D.J., Welbourn, W.C. (Eds.), Acarology IX Symposia. Ohio State University, Ohio, pp. 349–354. Klompen, J., Black IV, W.C., Keirans, J.E., Norris, D.E., 2000. Systematics and biogeography of hard ticks, a total evidence approach. Cladistics 16, 79–102. Leiby, D., 2011. Transfusion-transmitted Babesia spp.: bull’s-eye on Babesia microti. Clin. Microbiol. Rev. 24, 14–28. Lovisolo, O., Hull, R., Rösler, O., 2003. Coevolution of viruses with hosts and vectors and possible paleontology. Adv. Virus Res. 62, 325–379. Marrelli, M., Souza, L., Marques, R., Labruna, M., Matioli, S., Tonon, A., Ribolla, P., Marinotti, O., Schumaker, T., 2007. Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA. J. Med. Entomol. 44, 222–228. Mtambo, J., Madder, M., van Bortel, W., Berkvens, D., Backeljau, T., 2007. Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification. Exp. Appl. Acarol. 41, 115–128. Nava, S., Venzal, J., Labruna, M., Mastropaolo, M., González, E., Mangold, A., Guglielmone, A., 2010. Hosts, distribution and genetic divergence (16S rDNA) of Amblyomma dubitatum (Acari: Ixodidae). Exp. Appl. Acarol. 51, 335–351.
9
Parola, P., Paddock, C., Raoult, D., 2005. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin. Microbiol. Rev. 18, 719–756. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Pritt, B., Sloan, L., Johnson, D., Munderloh, U., Paskewitz, S., McElroy, K., McFadden, J., Binnicker, M., Neitzel, D., Liu, G., Nicholson, W., Nelson, C., Franson, J., Martin, S., Cunningham, S., Steward, C., Bogumill, K., Bjorgaard, M., Davis, J., McQuiston, J., Warshauer, D., Wilhelm, M., Patel, R., Trivedi, V., Eremeeva, M., 2011. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N. Engl. J. Med. 365, 422–429. Qi, C., Zhou, D., Liu, J., Cheng, Z., Zhang, L., Wang, L., Wang, Z., Yang, D., Wang, S., Chai, T., 2011. Detection of Babesia divergens using molecular methods in anemic patients in Shandong Province, China. Parasitol. Res. 109, 241–245. Shao, R., Barker, S., 2007. Mitochondrial genomes of parasitic arthropods: implications for studies of population genetics and evolution. Parasitology 134, 153–167. Shao, R., Campbell, N., Barker, S., 2001. Numerous gene rearrangements in the mitochondrial genome of the wallaby louse, Heterodoxus macropus (Phthiraptera). Mol. Biol. Evol. 18, 858–865. Shaw, M., Murrell, A., Barker, S., 2002. Low intraspecific variation in the rRNA internal transcribed spacer 2 (ITS2) of the Australian paralysis tick, Ixodes holocyclus. Parasitol. Res. 88, 247–252. Song, S., Shao, R., Atwell, R., Barker, S., Vankan, D., 2011. Phylogenetic and phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences. Int. J. Parasitol. 41, 871–880. Song, Z., Han, L., Zhao, H., Yan, D., Li, H., Zhang, L., Liu, S., Yue, L., 2004. Studies on epidemic disease of tick-borne encephalitis (TBE) surveillance and TBE virus isolated. Lett. Biotechnol. 15, 43–50. Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML Web servers. Syst. Biol. 75, 758–771. Sun, S., Aishan, M., Wang, X., Meng, W., Feng, C., Zhang, F., Hang, C., Hu, Z., Zhang, Y., 2009. Epidemiology and phylogenetic analysis of Crimean-Congo hemorrhagic fever viruses in Xinjiang, China. J. Clin. Microbiol. 47, 2536–2543. Swanson, S., Neitzel, D., Reed, K., Belongia, E., 2006. Coinfections acquired from Ixodes ticks. Clin. Microbiol. Rev. 19, 708–727. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tian, J., He, Y., Wei, P., Li, W., Jie, S.H., Yao, J.H., 2010. Retrospective analysis of clinical diagnosis of 21 cases of human ehrlichiosis. Chin. J. Infect. Dis. 28, 278–281. Tian, Z., Xie, J., Yin, H., Luo, J., Zhang, L., Zhang, P., Luo, J., 2011. Discrimination between Haemaphysalis longicornis and H. qinghaiensis based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2). Exp. Appl. Acarol. 54, 165–172. Uilenberg, G., Thiaucourt, F., Jongejan, F., 2004. On molecular taxonomy: what is in a name? Exp. Appl. Acarol. 32, 301–312. WHO, 2006. Lyme Borreliosis in Europe: Influences of Climate and Climate Change, Epidemiology, Ecology and Adaptation measures. World Health Organization, Regional Office for Europe, Copenhagen, Denmark. Yang, X., Chen, Z., Liu, J., 2008. The valid genus and species names of ticks (Acari: Ixodida: Argasidae, Ixodidae) in China. J. Hebei Norm. Univ. (Nat. Sci. Ed.) 32, 529–533. Zhang, L., Liu, Y., Ni, D., Li, Q., Yu, Y., Yu, X., Wan, K., Li, D., Liang, G., Jiang, X., Jing, H., Run, J., Luan, M., Fu, X., Zhang, J., Yang, W., Wang, Y., Dumler, J., Feng, Z., Ren, J., Xu, J., 2008. Nosocomial transmission of human granulocytic anaplasmosis in China. JAMA 300, 2263–2270. Zhang, Y.Z., Zhou, D.J., Xiong, Y., Chen, X.P., He, Y.W., Sun, Q., Yu, B., Li, J., Dai, Y.A., Tian, J.H., Qin, X.C., Jin, D., Cui, Z., Luo, X.L., Li, W., Lu, S., Wang, W., Peng, J.S., Guo, W.P., Li, M.H., Li, Z.J., Zhang, S., Chen, C., Wang, Y., de Jong, M.D., Xu, J., 2011. Hemorrhagic fever caused by a novel tick-borne Bunyavirus in Huaiyangshan, China. Zhonghua Liu Xing Bing Xue Za Zhi. 32, 209–220. Zhang, Y.Z., Zhou, D.J., Qin, X.C., Tian, J.H., Xiong, Y., Wang, J.B., Chen, X.P., Gao, D.Y., He, Y.W., Jin, D., Sun, Q., Guo, W.P., Wang, W., Yu, B., Li, J., Dai, Y.A., Li, W., Peng, J.S., Zhang, G.B., Zhang, S., Chen, X.M., Wang, Y., Li, M.H., Lu, X., Ye, C., de Jong, M.D., Xu, J., 2012. The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China. J. Virol. 86, 2864–2868.
Please cite this article in press as: Lu, X., et al., Molecular survey of hard ticks in endemic areas of tick-borne diseases in China. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.ttbdis.2013.01.003