Ticks and Tick-Borne Diseases 5 (2014) 864–870

Ticks and Tick-borne Diseases 5 (2014) 864–870

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Ticks and Tick-borne Diseases

j ournal homepage: www.elsevier.com/locate/ttbdis

Original article

The composition and transmission of microbiome in hard tick, Ixodes

persulcatus, during blood meal

a,1 b,1 a a

Xue-Chao Zhang , Zhang-Nv Yang , Bo Lu , Xiao-Fang Ma ,

a a,∗

Chuan-Xi Zhang , Hai-Jun Xu

Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China

Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310053, China

r t i c l e i n f o a b s t r a c t

Article history: The tick Ixodes persulcatus is the predominant tick species in Northeastern China, and it is a major vector

Received 19 March 2014

in transmission of tick-borne diseases. By 16S rRNA Illumina sequencing, we investigated the micro-

Received in revised form 3 June 2014

biome of I. persulcatus and assessed the variation of the microbiome before and after blood feeding. The

Accepted 4 July 2014

prolonged blood meal dramatically altered the composition of the microbiome but did not inﬂuence the

Available online 20 August 2014

bacterial diversity. Overall, 373 and 289 bacterial genera were assigned to unfed and fed ticks, respec-

tively. To investigate microbes that were potentially transmitted to vertebrate hosts during a blood meal,

Keywords:

we examined the microbiome in rat blood after tick bites. Our data showed that 237 bacterial genera

Ixodes persulcatus

Microbiome were suspected to be pathogens of vertebrates because they were commonly detected in unfed ticks, fed

ticks, and rat blood samples after tick bites. Additionally, the prevalence survey on Borrelia burgdorferi

Tick-borne pathogen

Blood meal s.l., Ehrlichia chaffeensis, Anaplasma phagocytophilum and Yersinia pestis was performed. We found that

16S ribosomal RNA B. garinii and B. afzelii are the predominant genospecies of the Lyme disease spirochete in I. persulcatus

ticks. This is the ﬁrst time that the microbial composition in this tick species and in rat blood transmitted

via tick bites has been reported. These data may ultimately assist in identiﬁcation of novel pathogens

transmitted by I. persulcatus ticks.

Introduction tick-borne diseases in humans in this region (Cao et al., 2003; Wan

et al., 1998).

Ticks are obligate, blood-sucking ectoparasites that are com- Ticks host a variety of disease-causing bacteria, including

monly found all around the world. They transmit a broad range Borrelia, Rickettsia, Anaplasma, Francisella, Coxiella and Ehrlichia

of infectious agents to humans and animals, including viruses, genera; and new pathogenic agents continue to be reported. For

bacteria and protozoa. They are considered to be second only to instance, a new member of the family Anaplasmataceae, Candida-

mosquitoes as worldwide vectors of human disease (de la Fuente tus Neoehrlichia mikuresis (Kawahara et al., 2004) has been found

et al., 2008; Jongejan and Uilenberg, 2004). The hard tick Ixodes per- in Ixodes ovatus ticks. This pathogen can cause illness that pre-

sulcatus is the most epidemiologically important vector from Russia dominantly develops in immunocompromised patients (Fehr et al.,

to eastern Asia, which is home to around one-ﬁfth of the world’s 2010; Welinder-Olsson et al., 2010). These ﬁndings provide unam-

population (Cao et al., 2000). Notably, this species is widely dis- biguous evidence that there are as-yet unidentiﬁed pathogens

tributed in Northern China and is most commonly responsible for associated with ticks. Additionally, co-infection of pathogens has

been recently reported in I. persulcatus ticks. This increases the

risk of multiple infections in humans, which could lead to more

severe clinical manifestations (Cao et al., 2003; Huang et al., 2006;

∗ Swanson et al., 2006). In contrast to the signiﬁcance of their medical

Corresponding author at: 866 Yu-Hang-Tang Rd., C1039 Nong-Sheng-Huan

Building, Zi-Jin-Gang Campus, Zhejiang University, Hangzhou 310058, China. and veterinary importance, the competency of ticks for pathogens

Tel.: +86 571 88982996; fax: +86 571 88982991. is largely unexplored (Parola and Raoult, 2001).

E-mail addresses: [email protected] (X.-C. Zhang),

Besides pathogens, ticks also harbor a variety of endosym-

[email protected] (Z.-N. Yang), [email protected] (B. Lu),

biotic bacteria that exist in commensal, mutualistic or parasitic

[email protected] (X.-F. Ma), [email protected] (C.-X. Zhang),

relationship with their arthropod hosts (Noda et al., 1997; Sacchi [email protected] (H.-J. Xu).

These authors contributed equally to this work. et al., 2004; Sassera et al., 2006; Scoles, 2004; Zhong et al., 2007).

http://dx.doi.org/10.1016/j.ttbdis.2014.07.009

X.-C. Zhang et al. / Ticks and Tick-borne Diseases 5 (2014) 864–870 865

Table 1

Multiple lines of evidence have demonstrated that interactions

Summary of sequencing results of unfed ticks, fed ticks and rat blood.

among microbes can affect pathogen fate and transmission (Clay

et al., 2008; de la Fuente et al., 2003; Macaluso et al., 2002; Unfed tick Fed tick Rat blood

Narasimhan et al., 2014). Hence, the biological importance of

Clean reads 119,200 55,833 197,043

microbial interactions within the tick vector together with the OTUs 14,098 8181 30,612

surveillance of well-known and emerging infectious agents associ- Shannon index 6.472 6.325 10.699

Good’s coverage 0.884 0.882 0.837

ated with I. persulcatus warrants the studies of bacterial diversity

present in this tick.

Given that most microorganisms that exist in nature are

V4 16S rRNA amplicon sequencing and data analysis

not amenable to culturing, classical culture-dependent methods

have only detected a fraction of the microbiome (Amann et al.,

The genomic DNA of individual unfed female adult ticks (n = 50),

1995). With the advance of molecular techniques, high-throughput

fed female adults (n = 10), or rat blood (n = 6) was combined at

sequencing technologies such as 16S rRNA amplicon sequencing

equimolar concentrations in three pools before 16S rRNA Illumina

or the shotgun metagenomic approach have been used to investi-

sequencing. The V4 region of the 16S rRNA gene was ampliﬁed

gate the entire array of bacteria associated with ticks under natural

with 515F-806R primers to construct the amplicon libraries accord-

conditions (Andreotti et al., 2011; Carpi et al., 2011; Nakao et al.,

ing to a previously described method (Peiffer et al., 2013). The

2013). The blood meal has been shown to increase bacterial diver-

paired-end sequencing was performed on an Illumina Miseq plat-

sity and induce bacteria multiplication in tick gut during the feeding

form (Novogene, Beijing, China) based on a standard protocol

process, suggesting that speciﬁc bacteria are more likely to be trans-

from the manufacturer. Raw data were screened and assembled

mitted to vertebrates (Azad and Beard, 1998; De Silva and Fikrig,

by QIIME (Caporaso et al., 2010) and FLASH (Magoc and Salzberg,

1995; Heise et al., 2010).

2011) software packages. The UCLUST method (Caporaso et al.,

In the present study, we conducted a comprehensive survey of

2010) was used to cluster the sequences into Operational Taxo-

the pathogen load and the microbiome associated with I. persulca-

nomic Units (OTUs) at an identity threshold of 97%. Meanwhile,

tus ticks before and after feeding via 16S rRNA amplicon Illumina

the RDP Classiﬁer (Wang et al., 2007) was used to assign each

sequencing. Also, we examined the microbiome in rat blood after

OTU to a taxonomic level. Additional analyses, such as rarefaction

tick bites to detect potential pathogens that are transmitted to

curves, Shannon index, and Good’s coverage, were carried out with

vertebrate hosts. We found that B. burgdorferi s.l. is the dominant

QIIME. In addition, the OTU table produced by the QIIME pipeline

bacterial pathogen in I. persulcatus, followed by A. phagocytophilum.

was imported into MEGAN 4 and mapped on the NCBI taxonomy

These results highlight the need for a thorough understand-

database (Huson et al., 2011). The genera shared among the three

ing of the medical and veterinary signiﬁcance of I. persulcatus

samples were represented by a Scale-Venn diagram using eulerAPE

ticks.

(http://www.eulerdiagrams.org/eulerAPE/). Metastats web inter-

face (White et al., 2009) was used to detect a statistically signiﬁcant

Materials and methods difference between unfed and fed tick microbiomes at phylum and

genus levels.

Ethics statement

PCR ampliﬁcation and sequence analysis

Experiments and animal care were conducted according to

protocols approved by the Institutional Animal Care and Use Com- The prevalence survey on B. burgdorferi s.l., E. chaffeensis, A.

mittee of Zhejiang University. phagocytophilum and Y. pestis was carried out via PCR, based on the

genomic DNA extracted from individual unfed male ticks (n = 50)

and unfed female ticks (n = 50). All primer sequences and gene tar-

Tick collection and storage

gets are listed in Table S1. The PCR products were separated using

1.5% agarose gel electrophoresis and visualized under UV light.

Host-seeking ticks were collected by dragging a white cloth over

Amplicons of the 5S-23S rRNA intergenic spacer of B. burgdorferi s.l.

vegetation in the woodland near Mudanjiang in May 2012 and

◦ ◦ were cloned with the pMD19-T Vector (TaKaRa, Dalian, China) and

2013 (N 44.916 , E 129.498 ), where B. burgdorferi and A. phagocy-

then subjected to conventional Sanger sequencing. The obtained

tophilum are known to be endemic (Cao et al., 2000). Field-collected

sequences were queried to the NCBI database to ﬁnd the closest

ticks were kept in conditional incubators. A subset of unfed I. per-

counterparts.

sulcatus adults (50 males and 50 females) were washed with 70%

ethanol via gently shaking for 30s. The ticks were then rinsed

Results

three times in sterile water to remove environmental contaminants

◦

and frozen at −80 C until use. To detect the microbiome in fed

Bacterial community shifts during the blood-feeding process

adults, unfed females were placed on naïve Sprague Dawley rats

(25 ticks/rat) until they were replete. Fed adults (10 females) were

The blood meal is critical for ticks to complete their life cycle and

surface-sterilized prior to genomic DNA extraction.

is also an important mechanism for pathogen transmission. In order

to investigate the inﬂuence of the blood meal on microbial compo-

Genomic DNA extraction sition, we surveyed the microbiome associated with I. persulcatus

ticks before and after feeding by 16S rRNA amplicon sequencing

Genomic DNA was extracted from individual unfed ticks (50 analysis. Our results showed that unfed ticks share a similar Shan-

males and 50 females) or fed ticks (10 females) using the QIAamp non diversity index with that of fed ticks (6.472 vs. 6.325) (Table 1).

DNA Micro Kit or QIAamp DNA Mini Kit according to the manufac- Rarefaction curves derived from the Shannon index indicated that

turer’s protocols (QIAGEN, Shanghai, China). The rat blood samples the plateau of diversity was achieved at around 20,000 reads, show-

were collected from the tail vein (6 rats) at one week after tick ing that our sequencing had adequate depth to capture diversity

infestation. DNA was extracted from each rat blood sample. The (Fig. S1A). Although the read number in the fed tick sample was

◦

−

extracted DNA was stored at 20 C until use. lower than that in the unfed tick sample, both samples showed

866 X.-C. Zhang et al. / Ticks and Tick-borne Diseases 5 (2014) 864–870

Fig. 1. Microbial composition in unfed ticks, fed ticks and rat blood. Phyla with a

Fig. 2. Scalar-Venn representation of shared genera among microbiome associated

relative abundance ≥0.1% were deﬁned as predominant phyla. Sequences that failed

with unfed ticks, fed ticks and rat blood. There were 373, 289 and 441 genera

to be classiﬁed or phyla with a relative abundance less than 0.1% were assigned as

assigned to unfed ticks, fed ticks and rat blood, respectively. Of these, 237 bacterial

‘Other’.

genera were detected in all samples and were suspected to be potential pathogens

to vertebrates.

similar Good’s coverage rates (0.884 vs. 0.882) (Table 1), indicat-

ing that the majority of the microbiome of I. persulcatus has been for this library indicated that a majority of the microbial species

detected. Increasing the sequencing depth could reveal additional were detected (Table 1). After taxonomic assignment, a total of 441

rare microbial species with fed ticks (Fig. S1B), but this would have genera were identiﬁed in the rat blood samples (Table S2). How-

little effect on the diversity estimation. ever, most microbes were sparsely represented; even the dominant

Subsequently, each OTU was assigned to a taxonomic level by genera only accounted for a low proportion of the bacterial commu-

the RDP Classifier, and most reads were classified to the Bacterial nity (between 0.5% and 5.3%) (Table 3). To find microbes that tend

and Archaeal kingdoms. The bacterial diversity and relative abun- to be transmitted to vertebrate hosts, we investigated the bacte-

dance of unfed and fed ticks are presented in Table S2. Overall, we rial genera that were commonly presented in unfed ticks, fed ticks,

assigned 373 and 289 genera to unfed and fed ticks, respectively. and rat blood samples. We identiﬁed 237 genera that were trans-

Considering a large amount of bacteria was categorized into mitted to the vertebrate host (Fig. 2 and Table S3), which made

the phylum Proteobacteria both in unfed and fed tick samples, up of 82% of genera detected in fed ticks. Statistical analysis was

≥

we therefore deﬁned phylum with relative abundance 0.1% performed to gain a better insight into the inﬂuence of the blood

as predominant phylum (Fig. 1). The different abundances of meal on these potential pathogens. We found that 50 of the 237

Acidobacteria, Actinobacteria, Bacteroidetes, Crenarchaeota, Firmi- genera propagated considerably after a blood meal while 22 of the

cutes, Nitrospirae, OP9, Proteobacteria, Spirochaetes and Tenericutes 237 genera were signiﬁcantly less abundant. Members of the phyla

between the two samples were statistically signiﬁcant (Table 2). Spirochaetes, Bacteroidetes and Proteobacteria largely accounted for

For instance, the proportions of Spirochaetes and Bacteroidetes in the signiﬁcant changes described. Members of these genera are

the fed tick samples were signiﬁcantly reduced. Sequences afﬁl- widespread in the environment. Some are pathogenic to humans,

iated with Borrelia within Spirochaetes and sequences afﬁliated and others are to animals. A number of them can induce clinically

with Chryseobacterium and Sphingobacterium within Bacteroidetes relevant opportunistic infections (de Bentzmann and Plesiat, 2011;

mainly contributed to this trend. Sequences afﬁliated with Acineto- Han and Andrade, 2005; Hsueh et al., 1997; Lambiase et al., 2009;

bacter, Rickettsia, Pseudomonas, and Brevundimonas dropped to low Michalopoulos and Falagas, 2010; O’Hara et al., 2000; Smith and

or undetectable levels after a blood meal (Tables S2 and S3). Their Gradon, 2003). Even some of the relatively less abundant genera,

places in relative abundance were taken by Proteus, Morganella, such as Ehrlichia and Yersinia, are also worth noting for the diseases

Comamonas within Proteobacteri. Despite the large variation in the that they cause. These results highlight the potential ability of I.

bacterial community during the blood meal, feeding had little or persulcatus to transmit various microbes. These ﬁndings also indi-

no impact on the microbial diversity associated with I. persulcatus cate that infectious agents beyond the most commonly reported

ticks. pathogens are harbored by ticks.

Potential bacterial pathogens transmitted to vertebrate hosts Prevalence of tick-borne pathogens

We then attempted to investigate the repertoire of bacteria In order to verify the prevalence of potential pathogens detected

transmitted to vertebrate hosts during blood feeding. Naïve rats by high-throughput sequencing, we examined the infectious ratio

were infected by unfed ticks (25 ticks/rat). One week post infec- of Borrelia burgdorferi s.l., Ehrlichia chaffeensis and Yersinia pestis in

tion, genomic DNA was extracted from whole rat blood for Illumina I. persulcatus adults collected in years 2012 and 2013. Given that

sequencing of 16S rRNA. A Shannon index revealed that the bacte- Anaplasma phagocytophilum is commonly reported in this endemic

rial diversity in rat blood samples was notably higher than that area, it was also included. The results revealed that of the 100 ticks

of unfed and fed ticks (Table 1 and Fig. S1A). The Good’s coverage examined, 43 were positive for B. burgdorferi s.l. (43%) and 5 were

X.-C. Zhang et al. / Ticks and Tick-borne Diseases 5 (2014) 864–870 867

Table 2

Differentially abundant phyla between unfed ticks (A), fed ticks (B) and rat blood (C).

Phylum A (%) B (%) C (%) A vs. B p-value

Acidobacteria 0.48 0.57 4.50 1.18E−02

Actinobacteria 1.53 1.87 6.42 1.75E−07

Bacteroidetes 7.63 3.10 19.12 6.35E−294

Chloroﬂexi 1.13 1.03 4.96 8.53E−02

Crenarchaeota 0.00 0.12 1.78 1.73E−33

Cyanobacteria 0.00 0.00 0.14 1.00E+00

Firmicutes 2.35 3.13 11.71 6.62E−22

Gemmatimonadetes 0.16 0.17 1.18 5.61E−01

Nitrospirae 0.00 0.14 0.27 5.95E−39

OP9 0.25 0.42 0.56 4.60E−09

Planctomycetes 0.27 0.23 1.86 1.50E−01

Proteobacteria 80.65 86.10 38.20 2.01E−171

Spirochaetes 0.51 0.00 0.12 4.25E−64

Tenericutes 1.66 0.00 0.13 4.68E−205

Thermi 0.00 0.00 0.13 1.00E+00

TM7 0.00 0.00 0.12 1.00E+00

Verrucomicrobia 0.29 0.31 3.17 3.33E−01

WPS-2 0.00 0.00 0.11 1.00E+00

WS3 0.00 0.00 0.14 1.00E+00

Other 3.10 2.79 5.37 4.05E−04

Table 3

Top 20 genera associated with unfed ticks, fed ticks and rat blood.

Unfed tick % Fed tick % Rat blood %

[1] Acinetobacter 21.58 Proteus 33.44 Halomonas 5.31

[2] Rickettsia 18.95 Rickettsia 4.92 Zhouia 4.93

[3] Pseudomonas 6.35 Morganella 2.29 Parapedobacter 3.30

[4] Chryseobacterium 2.82 Comamonas 1.94 Acinetobacter 2.46

[5] Sphingobacterium 1.20 Acinetobacter 1.88 DA101 1.81

[6] Brevundimonas 1.12 Halomonas 1.86 Ehrlichia 1.74

[7] Halomonas 0.93 Ehrlichia 0.78 Shewanella 1.58

[8] Proteus 0.79 Shewanella 0.75 Candidatus Nitrososphaera 1.40

[9] Zhouia 0.48 Corynebacterium 0.42 Bacteroides 1.00

[10] Borrelia 0.40 Prevotella 0.40 Proteus 0.94

[11] Prevotella 0.36 Pseudomonas 0.35 Solibacillus 0.75

[12] Parapedobacter 0.32 Zhouia 0.33 Ochrobactrum 0.70

[13] Clostridium 0.31 Clostridium 0.31 Sphingobacterium 0.69

[14] Flavobacterium 0.29 Ochrobactrum 0.27 Candidatus Solibacter 0.61

[15] Shewanella 0.29 Desulfotomaculum 0.25 Candidatus Xiphinematobacter 0.58

[16] Haemophilus 0.27 Chryseobacterium 0.22 Prevotella 0.56

[17] Janthinobacterium 0.26 Parapedobacter 0.22 Bacillus 0.54

[18] Psychrobacter 0.25 Polaromonas 0.22 Rhodoplanes 0.51

[19] Stenotrophomonas 0.18 Myroides 0.19 Sphingomonas 0.48

[20] Desulfotomaculum 0.18 Sphingomonas 0.16 Luteimonas 0.46

OTUs characterized to genera level were presented.

Table 4

Prevalence of tick-borne pathogens in I. persulcatus collected in Mudanjiang.

No. (%) of tick positive

Year Sex No. of ticks B.b.s.l. A.p. E.c. Y.p. B.b.s.l.&A.p.

2012 Male 26 8/26(30.8) 1/26(3.8) 0/26(0.0) 0/26(0.0) 0/26(0.0)

Female 26 12/26(46.2) 3/26(11.5) 0/26(0.0) 0/26(0.0) 3/26(11.5)

2013 Male 24 11/24(45.8) 1/24(4.2) 0/24(0.0) 0/24(0.0) 1/24(4.2)

Female 24 12/24(50.0) 0/24(0.0) 0/24(0.0) 0/24(0.0) 0/24(0.0)

Total 100 43/100(43.0) 5/100(5.0) 0/100(0.0) 0/100(0.0) 4/100(4.0)

B.b.s.l.: B. burgdroferi s.l., A.p.: A. phagocytophilum, E.c.: E. chaffeensis, Y.p.: Y. pestis.

positive for A. phagocytophilum (5%). However, neither E. chaffeensis The genospecies of B. burgdorferi s.l. in Northeastern China

nor Y. pestis was detected in ticks collected in either year (Table 4).

Accordingly, these data indicated that B. burgdorferi s.l. is the dom- Since B. burgdorferi s.l. is the dominant pathogen detected in

inant bacterial pathogen in ticks, followed by A. phagocytophilum, ticks, we determined its genospecies by sequencing the 5S-23S

while E. chaffeensis and Y. pestis are scarce in this region. Addition- rRNA intergenic region. PCR was performed on each B. burgdorferi

ally, we found that 4 ticks (4%) were co-infected with B. burgdorferi s.l.-positive tick (n = 43), and 43 amplicons that ranged in size from

s.l. and A. phagocytophilum, indicating an increased risk of simulta- 237 to 255 bp were generated. Based on BLAST alignment, the 43

neous human infection with these two pathogens. amplicons from B. burgdorferi s.l.-positive ticks could be clustered

868 X.-C. Zhang et al. / Ticks and Tick-borne Diseases 5 (2014) 864–870

into two genospecies: 13 ticks (30.2%) carried B. afzelii and 30 ticks (Narasimhan et al., 2014), and (c) rat tails were stringently sur-

(69.8%) carried B. garinii. These results, together with previous face sterilized during blood collection. We suspect that most of

reports (Chu et al., 2011; Takada et al., 1998), suggest that B. garinii the microbes would be quickly cleared by the rat immune system.

and B. afzelii are the two predominant genospecies of Borrelia in I. Nonetheless, some genera may be pathogenic to vertebrates under

persulcatus in Northeastern China. Moreover, we found that ticks certain conditions, for instance to immunocompromised patients.

could be co-infected with A. phagocytophilum and either B. afzelii or Also, some genera such as Yersinia and Chryseobacterium can induce

B. garinii, suggesting that more complicated clinical manifestations clinically relevant opportunistic infections. It has been reported

may exist in patients from this area. that Y. pestis can maintain and amplify in ticks for several years

(Thomas et al., 1990; Zykin, 1994), and this bacteria has been iso-

lated from Dermacentor nuttalli and Haemaphysalis verticalis ticks

B. garinii genospecies is infectious to vertebrates

collected at natural plague foci adjacent to Mudanjiang, China (Fang

et al., 2012; Liu et al., 2008; Tsow et al., 1959). Notably, it was the

Given that B. garinii and B. afzelii are the two main genospecies

ﬁrst time that Yersinia had been detected in I. persulcatus ticks in

of Borrelia in I. persulcatus ticks, we next asked whether they were

this area, although the species of this bacteria was not determined.

infectious to vertebrates. To this end, we infected naïve SD rats via

Three species of Yersinia (Y. pestis, Y. pseudotuberculosis and Y. ente-

tick bites (25 ticks/rat). One month after infection, ear punch biop-

rocolitica) have been exhaustively studied because of the human

sies were performed to detect spirochetes in the rats. All six rats

diseases that they cause (Bai et al., 2007; Drancourt, 2012; Pujol and

were positive for spirochete infection via visual inspection. Sub-

Bliska, 2005). There is much less knowledge about other species of

sequently, we ampliﬁed the 5S-23S rRNA intergenic region of the

Yersinia (Sulakvelidze, 2000). Some species of Yersinia have been

spirochetes recovered from the ear punch samples and found that

reported to account for human diarrheal infections (Noble et al.,

the B. garinii genospecies was transmitted to the rats through the

1987) and lethality in mice (Robins-Browne et al., 1991). This high-

tick bites. These data suggest that B. garinii might be the pathogenic

lights the need for further investigation into the biological function

Lyme genospecies in Northeastern China because it is capable of

of the Yersinia spp. found in I. persulcatus.

invading and establishing infection in vertebrate hosts via I. persul-

One caveat of this study is that the variation of microbial asso-

catus ticks.

ciation was only analyzed in unfed and fed ticks. The observed

changes in the bacterial community may not fully reﬂect the change

Discussion that occurs with a prolonged blood meal. Furthermore, a unique

microbiome may associate with different tick tissues (Andreotti

I. persulcatus is the predominant tick species in Northeastern et al., 2011). In addition, the Illumina sequencing described herein

China and is a major vector in transmission of tick-borne diseases was performed on pooled samples to eliminate the differences of

(Cao et al., 2003; Wan et al., 1998). Although epidemiologic surveys microbial associations caused by complex ecological factors and

have been extensively conducted recently, a comprehensive inves- maximize the likelihood of detecting potential pathogens. This

tigation of endosymbiotic bacteria in this species has been lacking. approach could decrease the sensitivity to detect low-copy bacteria

Herein, we performed Illumina sequencing of the 16S rRNA ampli- (Clay et al., 2008; Schabereiter-Gurtner et al., 2003) or reduce the

con to characterize the microbiome associated with I. persulcatus coverage of the microbial variability inside the population. Hence,

and assessed the variation of the microbiome before and after a it requires a ﬁner-scale sampling scheme and increased replicates

blood meal. Additionally, in order to detect potential pathogens in future studies.

that can be transmitted to vertebrate hosts, the microbiome in rat Two of the enzootic pathogens that associate with I. persulca-

blood after tick bites was examined for the ﬁrst time. tus, B. burgdorferi s.l and A. phagocytophilum, were detected in our

There are many factors can inﬂuence the bacterial community samples at prevalence rates comparable to levels shown in previ-

in ticks during a prolonged blood meal. For instance, nutritional ous reports in this area (Cao et al., 2003; Wan et al., 1998). Our

provisions differ in the gut of unfed and fed ticks, gut protein data, derived from animal model experiments, showed that the B.

expression changes in response to a blood meal (Anderson et al., garinii but not B. afzellii genospecies harbored by the I. persulca-

2008; Hajdusek et al., 2013), host immune molecules present in tus tick was capable of invading vertebrate hosts. Recently, the B.

a blood meal (Hajdusek et al., 2013), and the morphology and afzelii genospecies has been successfully isolated from a patient

function of the tick gut change in response to feeding (Grigor’eva, who resides in this area (Jiang et al., 2012), indicating that both

2003). Our results demonstrate that the composition of the I. B. garinii and B. afzellii genospecies are prevalent in Northeastern

persulcatus microbiome is dramatically altered during the pro- China. Accordingly, the heterogeneity and co-infection of the etio-

longed blood meal. However, the bacterial diversity remains at the logic agents of Lyme disease (Chu et al., 2011, 2008; Takada et al.,

same level before and after the feeding process. This observation 1998; Zhan et al., 2009), together with the diversity of clinical

conﬂicts with a previous report that characterized bacterial diver- manifestations, make diagnosis even more difﬁcult. Intriguingly,

sity in Amblyomma americanum through the construction of clone A. phagocytophilum failed to be detected by Illumina sequencing of

libraries (Heise et al., 2010). It was reported that the blood meal led 16S rRNA, which may partially be due to using pooled DNA samples

to an increase in bacterial diversity associated with A. americanum. for sequencing.

We suspect that this discrepancy may be due to the difference in Next-generation sequencing methods offer an efﬁcient

sensitivity between the two approaches as well as the tick species approach for exploring interactions among microbiomes, vec-

used. tors and their hosts under natural conditions. Through this

To detect potential pathogens that can be transmitted to verte- method, we showed the dramatic variation of the bacterial

brates, 16S rRNA sequencing was conducted using DNA extracted community in response to a blood meal and the repertoire of

from rat blood after tick bites. Surprisingly, 441 genera of microbes microbes that tend to be transmitted to vertebrates. Future studies

were detected in rat blood, of which 237 genera were shared by under a variety of controlled experimental conditions will be

blood samples that were collected after tick bites from unfed and needed to ﬁll in the signiﬁcant gaps in our knowledge of microbial

fed ticks. It is conceivable that the common microbes (237 gen- interactions with vectors and their hosts. Our present study

era) detected in blood samples were transmitted from the tick established a foundation for a comprehensive understanding of

bites because (a) they were detected in both the unfed and fed the function, ecology and evolution of I. persulcatus-associated

ticks, (b) blood of pathogen-free rats are supposed to be sterile microbiome.

X.-C. Zhang et al. / Ticks and Tick-borne Diseases 5 (2014) 864–870 869

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that they have no competing interests.

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