Bacterial Community in Haemaphysalis Ticks of Domesticated Animals

Home , Bartonella bovis, Coxiella (bacterium)

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Ticks and Tick-borne Diseases xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

journal homepage: www.elsevier.com/locate/ttbdis

Bacterial community in Haemaphysalis ticks of domesticated animals

from the Orang Asli communities in Malaysia

a,b a,b b a,b

Jing-Jing Khoo , Fezshin Chen , Kai Ling Kho , Azzy Iyzati Ahmad Shanizza ,

b a,b a,b a,b,∗

Fang-Shiang Lim , Kim-Kee Tan , Li-Yen Chang , Sazaly AbuBakar

Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya,

Kuala Lumpur 50603, Malaysia

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia

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

Article history: Ticks are vectors in the transmission of many important infectious diseases in human and animals. Ticks

Received 20 January 2016

can be readily found in the semi-forested areas such as the settlements of the indigenous people in

Received in revised form 8 April 2016

Malaysia, the Orang Asli. There is still minimal information available on the bacterial agents associated

Accepted 20 April 2016

with ticks found in Malaysia. We performed a survey of the bacterial communities associated with ticks

Available online xxx

collected from domestic animals found in two Orang Asli villages in Malaysia. We collected 62 ticks,

microscopically and molecularly identiﬁed as related to Haemaphysalis wellingtoni, Haemaphysalis hystri-

Keywords:

cis and Haemaphysalis bispinosa. Bacterial 16s rRNA hypervariable region (V6) amplicon libraries prepared

Microbiome

from the tick samples were sequenced on the Ion Torrent PGM platform. We detected a total of 392 pos-

Coxiella endosymbiont

sible bacterial genera after pooling and sequencing 20 samples, indicating a diverse bacterial community

Orang asli

Malaysia proﬁle. Dominant taxa include the potential tick endosymbiont, Coxiella. Other dominant taxa include

the tick-associated pathogen, Rickettsia, and environmental bacteria such as Bacillus, Mycobacterium,

Sphingomonas and Pseudomonas. Other known tick-associated bacteria were also detected, including

Anaplasma, Ehrlichia, Rickettsiella and Wolbachia, albeit at very low abundance. Speciﬁc PCR was per-

formed on selected samples to identify Rickettsia and Coxiella. Sequence of Rickettsia felis, which causes

spotted fever in human and cats, was identiﬁed in one sample. Coxiella endosymbionts were detected

in three samples. This study provides the baseline knowledge of the microbiome of ticks in Malaysia,

focusing on tick-associated bacteria affecting the Orang Asli communities. The role of the herein found

Coxiella and Rickettsia in tick physiology or disease transmission merits further investigation.

1. Introduction hosts life cycles, they are capable of supporting the transmission

of pathogens between hosts, in which humans frequently serve as

Ticks are excellent vectors for the transmission of zoonotic the accidental host. Multiple pathogenic agents may also be carried

infectious agents, including bacteria, viruses and protozoan par- by an individual tick, which could transmit these pathogens to the

asites, between human and animals. Ticks are known to harbor a human hosts during a bite (Carmichael and Fuerst, 2006). Ticks are

number of medically-important bacterial species within the Rick- also known to harbor endosymbionts or commensal bacteria. How-

ettsia, Anaplasma, Bartonella, Coxiella and Ehrlichia genera (Petney, ever, conventional methods for bacteria identiﬁcation, including

1993; Philippe et al., 2005). As ticks exhibit two-hosts or three- molecular or cultivation-dependent techniques, limits the detec-

tion of non-targeted or unexpected bacteria. With the advent

of next-generation sequencing (NGS), scientists are now able to

investigate the bacterial community carried by ticks, including

∗

Corresponding author at: Tropical Infectious Diseases Research and Education

unexpected bacteria that may have been missed using conventional

Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, Univer-

methods (Andreotti et al., 2011; Carpi et al., 2011; Menchaca et al.,

sity of Malaya, Kuala Lumpur 50603, Malaysia.

2013; Nakao et al., 2013; Vayssier-Taussat et al., 2013; Williams-

E-mail addresses: [email protected] (J.-J. Khoo),

[email protected] (F. Chen), [email protected] (K.L. Kho), Newkirk et al., 2014).

azzy [email protected] (A.I. Ahmad Shanizza), [email protected] Tick-borne illness, such as tick typhus and ehrlichiosis, in

(F.-S. Lim), [email protected] (K.-K. Tan), [email protected] (L.-Y. Chang),

humans or animals have been documented in Southeast Asian [email protected] (S. AbuBakar).

http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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2 J.-J. Khoo et al. / Ticks and Tick-borne Diseases xxx (2016) xxx–xxx

countries (Petney, 1993; Sirisanthana et al., 1994; Sagin et al., 2000; inants and also protection of researchers from potential infectious

Tay et al., 2000; Irwin and Jefferies, 2004). Most of the ﬁndings were pathogens. Tick samples were ﬁrst pulverized in liquid nitrogen

based on serological or microscopic examinations of blood samples using chilled mortar and pestle, using one set for each sample.

from humans or animals, such as domestic dogs or cats, presented Before use, all mortars and pestles were soaked in 10% sodium

with symptoms of tick-borne illnesses. Molecular approaches such hypochlorite solution for an hour, rinsed with sterile deionized

◦

as polymerase chain reaction (PCR) have also led to the identi- water and baked at 160 C overnight to eliminate contaminat-

ﬁcation of pathogenic bacteria, including Rickettsia, Ehrlichia and ing materials as much as possible. The resulting ﬁne powder was

Anaplasma in ticks sampled from forests or villages in Thailand, resuspended in 500 ␮L of sterile phosphate-buffered saline (PBS).

Philippines, Laos and Malaysia (Hirunkanokpun et al., 2003; Parola An aliquot of the suspension (200 ␮L) was used for DNA extrac-

et al., 2003a,b; Kernif et al., 2012; Ybanez et al., 2013; Kho et al., tion using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany)

2015a,b). according to manufacturer’s protocol. DNA was eluted in 100 ␮L

In Malaysia, the indigenous people, also known as the Orang Asli, of Ultrapure DNA/RNA-free distilled water (Invitrogen Life Tech-

live in remote villages or settlements within or nearing the trop- nologies, MA, USA). Mock extraction was performed in parallel

ical rainforests in the Malay peninsula or the Borneo island. The using the same lot of reagents. DNA concentration was determined

livelihood of most of these communities is still very much depen- by spectrophotometry using the Implen Nanophotometer (Implen,

dent on the hunting and foraging activities within the forests (Khor Munich, Germany). Molecular identiﬁcation of the tick samples

and Zalilah, 2008). The Orang Asli people also live in close contact were performed using previously published primers for the ampli-

with domestic animals such as dogs, cats or chickens, which they ﬁcation of the mitochondrial 16 rRNA partial sequence (Black and

keep as companion animals or as livestock. As the lifestyle of the Piesman, 1994).

Orang Asli involves frequent encounters with wildlife or domestic

animals, which are common hosts for ticks, the Orang Asli people 2.3. Preparation of the barcoded 16s rRNA V6 amplicon libraries

are at higher risk of contracting accidental tick bites and tick-

borne illnesses. There has only been one earlier study investigating The presence of bacteria in the tick samples were detected via

the seroprevalence of tick-borne rickettsial infections within the nucleic acid ampliﬁcation using barcode-tagged primers targeting

aboriginal communities in Sarawak, Malaysia (Sagin et al., 2000). the V6 hypervariable region of the 16s rRNA gene (nt 872–1052 of

Pathogen detection in ticks from Malaysia has only been reported in the complete 16s rRNA sequence of Escherichia coli, NCBI GenBank

a handful of studies, which identiﬁed the presence of spotted fever Accession: AJ605115) as previously described (Carpi et al., 2011).

group rickettsial species and Coxiella from livestock and domes- The V6 region allows for most discrimination between pathogenic

tic animals (Tay et al., 2014; Kho et al., 2015a,b; Koh et al., 2015; and non-pathogenic bacteria (Carpi et al., 2011). The forward

Watanabe et al., 2015). Hence, more studies are necessary to deter- primers consisted of the A-adaptor sequence (Ion Torrent-speciﬁc

mine the presence and risks of tick-borne diseases in this region, sequence), an Ion Xpress barcode sequence (Life Technologies,

especially within the communities of the Orang Asli. MA, USA) and a GAT linker fused to the 5 end of the 16s rRNA

In this study, we sampled ticks found feeding on domestic ani- target sequence. Each of the 20 tick samples was assigned to 1

mals kept by the Orang Asli from two villages in Malaysia. We of the 20 barcode sequences (Ion Xpress 77–96) used for sam-

investigated the bacterial community harbored by these ticks by ple identiﬁcation and demultiplication during sequence analysis.

using the 16s rRNA V6 hypervariable region amplicon sequencing The reverse primer consisted of a CC linker and the Pi-adaptor

approach utilizing the Ion Torrent PGM platform technology. sequence (trP1, Ion Torrent-speciﬁc sequence) fused to the 5 end of

the 16s rRNA target sequence. For the generation of the V6 ampli-

con library, DNA ampliﬁcation was performed in 50 ␮L reaction

2. Material and methods

mixture, containing 2 ␮L of extracted DNA, 2.5 units of Dreamtaq

DNA polymerase (Thermo Scientiﬁc, MA, USA), 0.2 ␮M of dNTPs

2.1. Tick samples

(Promega, WI, USA), and 0.2 ␮M each of the forward and reverse

primers. PCR was performed with the following conditions: acti-

Feeding ticks were collected from domestic animals (9 dogs, 6 ◦

vation at 94 C for 3 min, followed by 32 cycles of denaturation at

cats and 5 chickens) observed in 2 separate villages of Orang Asli ◦ ◦ ◦

94 C for 1 min, annealing at 56 C for 1 min and extension at 72 C

in the Perak state of Malaysia. All ﬁeld sampling activities were ◦

for 2 min, with a ﬁnal elongation step at 72 C for 2 min. The result-

performed with the approval from the Department of Orangs Asli

ing amplicons were separated by 1.5% agarose gel electrophoresis

Development, Malaysia (JAKOA). Once removed from the hosts, ®

stained with the SYBR Safe nucleic acid stain (Invitrogen Life Tech-

the ticks were quick-frozen in liquid nitrogen for transportation

◦ nologies, MA, USA). All amplicons observed with the expected DNA

and stored in −80 C until further processing. The collected ticks

molecular weight were gel puriﬁed using the QIAquick Gel Extrac-

were microscopically identiﬁed to the genus level and classiﬁed

tion Kit (Qiagen, Hilden, Germany) following the manufacturer’s

by life stage according to published taxonomic key for Haema-

protocol.

physalis ticks (Tanskul and Inlao, 1989). The adult ticks were all

In parallel, negative control PCR reactions were set up using

identiﬁed as female ticks. Adult ticks were processed as individ-

only Ultrapure DNA/RNA-free distilled water (Invitrogen Life Tech-

ual samples, while nymphs and larvae were pooled into groups of

nologies, MA, USA) in place of DNA samples. PCR reactions were

3–10 depending on the size (Table 1). For DNA extraction, frozen

also performed with the pre-homogenization water washes and

tick samples were thawed, washed thrice in 70% ethanol followed

elutions from mock DNA extractions as described above. These

by sterile deionized water to remove environmental debris and

reactions did not produce any detectable amplicons upon evalu-

disinfect the surface (Carpi et al., 2011). Water washes were sub-

ation with gel electrophoresis and hence excluded from further

jected to nucleic acid ampliﬁcation using bacterial-speciﬁc primers

study.

as described below.

2.4. Sequencing on the Ion Torrent PGM platform

2.2. DNA extraction from tick samples

Prior to clonal ampliﬁcation, concentrations of each ampli-

DNA extraction procedures were performed in a biosafety con were determined using Qubit dsDNA HS assay kit (Life

cabinet to ensure sample protection from environmental contam- Technologies, MA, USA). To prepare the DNA libraries, ampli-

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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Table 1

Haemaphysalis tick samples used for the preparation of V6 amplicon sequencing in this study.

Tick sample number Location Host Life stage Number of ticks pooled Blastn results for partial % Identity

mitochondrial 16s rRNA sequence

(NCBI accession number)

BdA1 Village B Dog Adult 1 H. bispinosa (KC853420.1) 99%

BdA2 Dog Adult 1 H. bispinosa (KC853419.1) 99%

BdA3 Dog Adult 1 H. hystricis (AB819198.1) 97%

BdA4 Dog Adult 1 H. hystricis (AB819198.1) 97%

BdN1 Dog Nymph 3 H. bispinosa (KC853419.1) 100%

BdN2 Dog Nymph 5 H. wellingtoni (AB819221.1) 100%

BdL1 Dog Larva 5 Not amplifed

BcN1 Cat Nymph 3 H. wellingtoni (AB819221.1) 100%

BcL1 Cat Larva 10 H. wellingtoni (AB819221.1) 100%

BchN1 Chicken Nymph 1 H. wellingtoni (AB819221.1) 100%

PdA1 Village P Dog Adult 1 H. hystricis (AB819197.1) 100%

PdA2 Dog Adult 1 H. hystricis (AB819197.1) 99%

PcN1 Cat Nymph 3 H. wellingtoni (AB819221.1) 100%

PcN2 Cat Nymph 4 H. wellingtoni (AB819221.1) 100%

PcL1 Cat Larva 5 H. wellingtoni (AB819221.1) 97%

PcL2 Cat Larva 5 H. wellingtoni (AB819221.1) 99%

PchA1 Chicken Adult 1 H. wellingtoni (AB819221.1) 100%

PchN1 Chicken Nymph 1 H. wellingtoni (AB819221.1) 100%

PchN2 Chicken Nymph 5 Not ampliﬁed

PchL1 Chicken Larva 5 H. wellingtoni (AB819221.1) 100%

d: dog, c: cat, ch: chicken, A: adult, N: nymph, L: larva.

cons of equal concentration for the same sample were pooled 2.6. Alpha rarefaction

and adjusted to the ﬁnal concentration of 8–12 pM. Emulsion

PCR was carried out with Ion OneTouchTM 200 Template Kit Sequence alignment was performed using the PyNAST (1.2.2)

V2 DL (Life Technologies, USA) according to the manufacturer’s alignment method. The aligned sequences were used to build a

protocol PNMAN0007220 Rev. 4.0. The ampliﬁed Ion Sphere Par- phylogenetic tree using FastTree (Price et al., 2009). The result-

ticles were enriched using Dynabeads MyOne Streptavidin C1 ing tree was used to generate alpha-rarefaction plots, with random

beads (Life Technologies, MA, USA) and enrichment efﬁciency was subsampling performed at incremental steps of 1000 reads and 10

assessed using the Ion Sphere Quality Control Kit (Life Technolo- iterations at each step.

gies, MA, USA). The sequencing of the amplicons were performed

using Ion Sequencing 200 kit (Life Technologies, MA, USA) as

2.7. Beta diversity

previously described (Tan et al., 2015). A total of 20 barcoded

samples were pooled and loaded into a single 316 chip (Life

Beta diversity was calculated using the unweighted and

Technologies, MA, USA). All resulting sequences were deposited

weighted UniFrac metric, followed by Principle Coordinates Anal-

into the European Nucleotide Archive (ENA, Study Accession:

ysis (PCoA) using the FastUniFrac workﬂow (Hamady et al., 2010).

PRJEB9707).

Three dimensional PCoA plots were generated using EMPeror

implemented in QIIME (Vázquez-Baeza et al., 2013).

2.8. PCR screening of selected bacteria

2.5. Sequence analysis

PCR ampliﬁcation of the partial Rickettsia gltA and Coxiella 16s

rRNA genes were performed using previously described primers

Demultiplexed and quality-ﬁltered reads from the Ion

and ampliﬁcation protocols to detect Rickettsia (Kho et al., 2015a,b)

Torrent Suite (Version 4.0.2) software were converted into

and Coxiella (Duron et al., 2014) respectively. PCR amplicons were

FASTA and quality ﬁles using SAMtools (Version 0.1.19,

electrophoresed and extracted as described above. Sequencing of

http://samtools.sourceforge.net/). Results were then analyzed

the amplicons was performed by a third party service provider

using the QIIME workﬂow (MacQIIME version 1.8.0-20140103)

(First Base Laboratories, Malaysia). The resulting sequences were

(Caporaso et al., 2010). Firstly, all resulting FASTA ﬁles were

compared to the available sequences in the NCBI GenBank database

combined and relabeled to ensure compatibility with the QIIME

(http://www.ncbi.nml.nih.gov/BLAST/).

workﬂow. Adaptor and primer sequences were removed from

the sequences, allowing for a maximum of 2 nucleotides of

primer mismatches. Quality ﬁltering were applied using default 3. Results

parameters in QIIME, except to only retain sequences between

125 nt to 220 nt in length. Filtered sequences were subjected 3.1. Tick samples

to de novo operational taxonomic unit (OTU) picking using the

UCLUST method at 97% similarity. Taxonomic assignment was In total, 20 distinct samples were used for sequencing, each

performed using RDP Classiﬁer (Version 2.2) with conﬁdence value consisting of individual adult, female ticks or pools of nymphs

of 50% based on the Greengenes reference database (Version 13 8). or larvae collected from dogs, cats, or chickens as indicated in

ChimeraSlayer (Haas et al., 2011) was used to remove chimeric Table 1. Molecular identiﬁcation demonstrated 11 samples highly

sequences from the OTU Table Singletons were also removed from identical (97–100%) to Haemaphysalis wellingtoni (NCBI Accession:

the OTU table before rarefaction to minimize the inﬂation of the AB819221.1), 4 samples similar (97–100%) to Haemaphysalis hystri-

number of detected OTUs due to potential PCR bias and sequencing cis (NCBI Accession: AB819198.1), and 3 samples similar (99–100%)

error. to Haemaphysalis bispinosa (NCBI Accession: KC853420.1, Table 1).

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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2000

1800

1600

1400 ies

ec 1200

1000 d sp

800 serve 600 Ob

400

200

0 5000 10000 15000 20000 25000 30000 35000 40000

Numb er of Reads

Bd A1 BdA2 BdA3 BdA4 BdN1 BdN2 BdL1

BcN1 BcL1 BchN1 PdA1 PdA2 PcN1 PcN2

PcL1 PcL2 PchA1 PchN1 PchL1

Fig. 1. Rarefaction curves of Haemaphysalis ticks bacterial 16s rRNA amplicon sequences. Quality-ﬁltered 16s rRNA amplicon sequences were subjected to alpha rarefaction

up to 40,000 sequences per sample.

We were unable to detect ampliﬁed DNA fragments from BdL1 and (see Table S.1), with nymphs exhibiting higher number of domi-

PchN2 despite repeated attempts. nant taxa (average of 18 per sample) compared to adults or larvae

(both at anaverage of 7 per sample).

PCoA plot based on the unweighted Unifrac distance showed

3.2. 16s rRNA V6 amplicon sequencing results

possible clustering of H. wellingtoni and H. hytricis samples respec-

tively, suggesting more similar bacterial community shared by

In this study, sequencing the amplicons of the V6 hypervariable

samples of the same tick species (Fig. 3A). H. bispinosa samples

region of the bacterial 16s rRNA gene on the Ion Torrent PGM plat-

showed less deﬁned clustering, with possible overlap with H.

form produced a number of reads ranging from 66118 to 147221

wellingtoni and H. hytricis samples. The sole Haemaphysalis sp. sam-

for 19 out of the 20 ticks samples tested (see Table S.1). PchN2

ple did not cluster with other tick species. Since the three life stages

was omitted from further analysis due to its small number of reads

were only represented in the H. wellingtoni samples, PCoA plot

(424). Initial quality ﬁltering step in the QIIME workﬂow yielded

was generated according to life stage for these samples (Fig. 3B).

54640–126559 sequences (82.6% to 92.5% of raw sequences) for

There was no clear clustering of samples based on the different life

downstream analysis. To overcome the possible effects of unequal

stages. PCoA plot based on weighted Unifrac distances did not show

sequencing output across samples, the sequences were rareﬁed to

deﬁned clusterings of samples based on species of life stage (data

40,000 reads for use in OTU picking and taxonomic assignment

not shown).

(Ponnusamy et al., 2014).

3.3. Bacterial diversity in Haemaphysalis tick samples 3.4. Bacterial taxa detected in Haemaphysalis tick samples

After rarefaction, a total of 787 bacterial taxa (see Table S.2) A number of bacterial genera commonly associated with ticks

were identiﬁed for the 19 samples, with 392 taxa assigned to the were observed in these samples. Coxiella accounted for the most

genus level. Each sample exhibited the number of taxa in the range abundant bacterial taxa (39.2%) in all samples collectively (Table 2).

of 200–334 (see Table S.1). The rarefaction curves each of the tick Coxiella was detected in all 19 samples, ranging from 0.23% to

samples approached saturation at 40,000 reads (Fig. 1), indicating 89.38% in each sample (Table 3), with a number of nymphs and lar-

sufﬁcient sampling depth. vae samples exhibiting lower relative abundance (BdL1 and BcN1).

At the class level, taxonomical composition of the 19 tick Rickettsia was the second most abundant bacterial taxa collectively

samples indicated that the dominant bacterial classes in the sam- (6%, Table 2). Rickettsia was observed in 16 out of 19 samples, rang-

ples are Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, ing from 0.01% to 55.1% of the total reads in each sample (Table 3).

Bacilli and Deltaproteobacteria, in combination representing 80% to Speciﬁcally, Rickettsia was highly abundant in 3 samples, namely

99% of the population in each of the sample (Fig. 2). Only 10 bacte- BdN2 (37.12%), BdL1 (55.1%), and PcL2 (15.95%).

rial taxa were most represented (≥1%) for the 19 samples combined Anaplasma and Ehrlichia were detected in 3 out of 19 samples,

(Table 2), in which 8 out of 10 bacterial taxa were assigned to the albeit at low abundance (less than 1%, Table 3). BcN1 exhibited the

genus level. Collectively, 0.97% of the reads in the 19 samples were highest level of Anaplasma (0.18%) and Ehrlichia (0.16%) out of the

unassigned to any taxon beyond the bacteria domain. In each sam- 3 samples detected. Although not assigned to the genus level, PcN1

ple, the number of dominant taxa (≥1%) ranged from 2 to 24 taxa displayed approximately 10% reads assigned to Anaplasmataceae

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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Fig. 2. Relative abundance of 5 major taxa at the class level in the 19 Haemaphysalis tick samples.

Table 2

Assigned taxa with abundance >1% of total population in tick samples as identiﬁed by QIIME.

Assigned taxa Abundance (%)

k Bacteria;p Proteobacteria;c Gammaproteobacteria;o Legionellales; f Coxiellaceae;g Coxiella 39.2

k Bacteria;p Proteobacteria;c Alphaproteobacteria;o Rickettsiales; f Rickettsiaceae;g Rickettsia 6.0

k Bacteria;p Proteobacteria;c Alphaproteobacteria;o Rhizobiales; f Phyllobacteriaceae;g 4.7

k Bacteria;p Firmicutes;c Bacilli;o Bacillales;f Bacillaceae; g Bacillus 3.8

k Bacteria;p Proteobacteria;c Gammaproteobacteria;Other;Other;Other 2.9

k Bacteria;p Actinobacteria;c Actinobacteria;o Actinomycetales; f Mycobacteriaceae;g Mycobacterium 2.8

k Bacteria;p Proteobacteria;c Alphaproteobacteria;o Sphingomonadales;f Sphingomonadaceae;g Sphingomonas 1.7

k Bacteria;p Proteobacteria;c Gammaproteobacteria;o Pseudomonadale;f Pseudomonadaceae;g Pseudomonas 1.5

k Bacteria;p Proteobacteria;c Alphaproteobacteria;o Rhizobiales; f Methylobacteriaceae;g Methylobacterium 1.3

k Bacteria;p Firmicutes;c Bacilli;o Bacillales;f Staphylococcaceae; g Staphylococcus 1.3

k: kingdom; p: phylum; c: class; o: order; f: family; g: genus.

(see Table S.2), indicating the presence of an unclassiﬁed bacteria to humans or animals, but have limited information on the role

potentially similar to the other members of Anaplasmataceae. Bar- of ticks in disease transmission. Mycobacterium, Pseudomonas

tonella, another group of tick-associated pathogen, was detected at and Staphylococcus were amongst the 10 most abundant taxa

very low abundance (0.003% to 0.03%) in 5 samples (Table 3). These in the 19 samples collectively, at 2.8%, 1.5% and 1.3% respec-

bacteria were detected together with the presence of Rickettsia tively (Table 2). These bacterial taxa were observed in all samples

in the same samples, indicating possible coinfection. Other well (Table 3). Examples of other less abundant bacterial taxa detected

studied tick-associated pathogens, Borrelia and Francisella, were include Acinetobacter (19/19, positive/total samples), Corynebac-

not detected in these samples. Wolbachia and Rickettsiella, which terium (19/19), Streptococcus (18/19), Stenotrophomonas (18/19),

are possible endosymbionts in ticks apart from Coxiella, were also Clostridium (13/19), Klebsiella (8/19), Serratia (3/19) and Leptospira

detected in these tick samples. Rickettsiella was observed in 16 (3/19) (see Table S.2). Bacteria normally associated with the envi-

samples at low abundance (0.01% to 0.58% in each sample). Wol- ronment or the soil were also detected in our samples. Speciﬁcally,

bachia was highly represented (8.4%) in 1 sample, BdA4, while being Bacillus, Sphingomonas and Methylobacterium, which were previ-

detectable at low abundance (0.01% to 0.18%) in additional 5 sam- ously reported to be detected in ticks, were detected in all samples

ples. studied (Tables 2 and 3).

There was also numerous bacterial genera identiﬁed in our tick

samples, which consists of species that are potentially pathogenic

Fig. 3. PCoA plots.PCoA plots based on unweighted Unifrac distances according to (A) tick species (red: H. bispinosa, blue: H. hystricis, green: H. wellingtoni and yellow:

Haemaphysalis sp.) and (B) life stage of H. wellingtoni samples (green: adult, red: nymph and blue: larvae. (For interpretation of the references to colour in this ﬁgure legend,

the reader is referred to the web version of this article.)

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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3.5. PCR detection of Rickettsia and Coxiella samples

Out of the 3 samples with the highest abundance of Rick-

ettsia (BdN2, BdL1, and PcL2), we successfully ampliﬁed and positive

sequenced the partial gltA gene from BdL1 (NCBI Genbank Acces- of

sion: KU948227), in which the sequence was 100% identical to

Rickettsia felis (NCBI Accession: JN375500.1). Sequencing of the gltA

3 3 5 6

gene fragment from PcL2 generated less than satisfactory results

Number 16 19 16 19 19 19 19 19 19

despite multiple attempts. No ampliﬁcation was observed for BdN2.

Hence, the Rickettsia strain in PcL2 and BdN2 could not be conﬁrmed

0 0 0 0 0.08 0 0.16 0.50 0.82 in this study. The partial 16s rRNA sequences for Coxiella were suc- PchL1 7.96 2.08 1.49

58.55

cessfully ampliﬁed from 5 (ENA Accession: LT548048–LT548052)

out of all 19 samples tested. The Coxiella strains in BdN2 (96%),

0.18 0 0 0 0.01 0 0.50

BcL1 (96%), PdA1 (97%) and PcL1 (96%) were closely similar to PchN1 8.84 1.33 2.80 3.74 5.33 1.30

the Haemaphysalis longicornis Coxiella symbiont (NCBI Accession:

AY342035.1). The Coxiella strain in BdA2 was highly similar (96%)

0.03 0 0 0 0.01 0.07 0.26 0.07 0.44 0.05 0.18 0.24

PchA1 89.38

to the Coxiella symbiont in Haemaphysalis shimoga (NCBI Accession:

HQ287535.1).

0.005 0 0.003 0.02 0.03 0.39 0.28 0.07 0.06 0.42 0.19 PcL2 15.95 73.70

4. Discussion

0.01 0 0 0 0.03 0 0.30 0.21 0.03 0.28 0.29 PcL1 2.98

61.07

In this study, we present a survey of the bacterial commu-

nity in Haemaphysalis ticks in Malaysia obtained using the NGS

0.47 0 0 0 0.58 0.18 0.66

2.98 1.83 1.25 2.31 5.22

technology. These ticks were sampled from the domestic ani- PcN2 12.29

mals (dogs, cats and chickens) observed in villages of the local

indigenous people, the Orang Asli, from two different locations in

0.07 0 0 0 0.16 0 0.43 0.03 PcN1 5.48 1.01 1.44 2.56

47.43

Malaysia. Haemaphysalis ticks are the dominant ticks in our sam-

pled sites, consisting of species identical or related to H. wellingtoni,

0.88 0 0 0 0 0.01 0.15 0.06 0.04 0.31 0.08 0.03 H. hystricis and H. bispinosa. This observation is consistent with pre- PdA2

86.16

vious reports suggesting the abundance of Haemaphysalis ticks in

Malaysia (Audy et al., 1960; Paramasvaran et al., 2009) and also

0 0 0.02 0 0.04 0 0.09 0.48 0.82 0.13 0.19 0.05

PdA1

the prevalence of these ticks in domestic animals in South East 78.99

Asia (Irwin and Jefferies, 2004). Ticks of the Haemaphysalis genus

had been implicated as potential disease vector to humans and

0.01 0 0 0 0.17 0 0.89

BchN1 1.08 4.40 1.64 6.55 1.00

animals worldwide. Various pathogenic bacteria have been previ- 16.36

ously detected in Haemaphysalis ticks including the disease agents

ticks

0.64 0 0 0 0.01 0 0.42 0.19 for rickettsial spotted fever, tick typhus, anaplasmosis and ehrli- BcL1 1.41 2.00 2.26 1.66 61.20 in

chiosis (Hirunkanokpun et al., 2003; Liu et al., 2013; Tijsse-Klasen

et al., 2013; Yu et al., 2015).

status 0.10 0.18 0.16 0.03 0.23 0.13 0 0.43 0.82 BcN1 9.49 1.24 5.93 6.56

In total, 392 bacterial taxa were identiﬁed to genus level in our

study. Although not directly comparable, metagenomics sequenc-

0 0 0 0.26 0.05 0 0.44 0.29 0.05 ing in previous reports, which sequenced full genomic DNA, yielded BdL1 1.67 3.08 1.32

55.10

138 and 156 genera respectively in Haemaphysalis formosensis and

transmission

H. longicornis (Nakao et al., 2013). Microbiome studies performed

0 0 0 0.02 0 0.43 0.91

BdN2 4.64 1.84 2.46 1.11 1.11

on other tick species revealed a variation in numbers of bacte- 37.12

rial genus identiﬁed. Carpi et al. (2011), who targeted the same

unknown

V6 region of the 16s rRNA gene, detected 108 bacterial genera in 0.48 0 0 0.003 0.01 0.06 0.62 0.72

BdN1 8.52 5.18 1.22 1.43 2.17

Ixodes ricinus. The cattle tick, Rhipicephalus microplus, was found but

sample.

to harbor 121 bacterial genera (Andreotti et al., 2011). In Ambly-

tick

0.86 0.003 0.003 0.02 0.03 0.45 0.70 omma americanum, two separate studies found 237 (Ponnusamy 8.4 1.24 1.73 5.93 2.34

BdA4 27.07

each et al., 2014) and 99 (Williams-Newkirk et al., 2014) genera, respec- members

tively. The vast difference in the numbers of genera may be due (%)

0 0 0.01 0.14 0 0.94 0.12 0.07 0.58 0.16

1.28

to the variations in the sequencing platform technology or ana- BdA3 29.82 35.12 taxa

lytical procedures employed. In addition, the high number of

ticks

pathogenic

bacterial genera may include bacterial species that were found in

0 0 0 0 0 0 0.47 0.13 0.97 0.29 BdA2 1.58 6.54 abundance

bacterial

64.65 on the surface of the tick samples, which may have originated

from the environment such as the soil or the skin surface of the

bacteria 0.08 0 0 0 0 0 0.55

3.97 1.60 2.26

host mammal despite our efforts in washing the sample. Previ- potentially Relative BdA1 16.19 15.36 16.36

selected

ous studies have demonstrated a substantial difference in bacterial of

bacteria with

community composition between surface-sterilized (DNA removed

with sodium hypochlorite) and non-sterilized A. americanum tick taxa

samples (Menchaca et al., 2013). Since bacterial taxa such as Bacil-

abundance

lus, Clostridium, Methylobacterium, Mycobacterium, Pseudomonas, 3

Sphingomonas and Staphylococcus, some of which were found in Samples Medically-important Rickettsia Bacillus Mycobacterium Anaplasma Rickettsiella Wolbachia Pseudomonas Staphylococcus Environmental Sphingomonas Methylobacterium Ehrlichia Bartonella Endosymbionts Coxiella Abundant Table

Relative

high abundance in our samples, comprise of species that may be

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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J.-J. Khoo et al. / Ticks and Tick-borne Diseases xxx (2016) xxx–xxx 7

commonly associated with the environment or as part of the mam- We observed Coxiella in our samples, which was also detected

malian skin ﬂora, it is possible that these bacteria orginated from in a previous study of Haemapysalis ticks (Nakao et al., 2013). Due

the external surfaces of the ticks in our study. More stringent to the short sequence of the 16s rRNA V6 region used in NGS, we

washing procedure, such as washing with sodium hypochlorite, or were unable to differentiate between Coxiella endosymbionts or

limiting sampling to internal organs such as the salivary glands and Coxiella burnetii. However, PCR detection based on longer but par-

mid-gut, may be necessary to determine the internal ﬂora of ticks tial 16s rRNA sequences ampliﬁed from 5 samples indicated the

in further studies. presence of Coxiella symbionts closely similar to previously identi-

A number of studies have reported distinct bacterial commu- ﬁed Coxiella endosymbionts in H. longicornis (Lee et al., 2004) and H.

nity structure based on tick species and life stage for Dermacentor shimoga (Ahantarig et al., 2011). Our study reports the presence of

variabilis, Dermacentor reticulatus, Ixodes scapularis, Ixodes persul- Coxiella 16s rRNA partial sequence in ticks related to H. wellingtoni,

catus and Ixodes pavlovskyi respectively (Hawlena et al., 2013; H. hystricis or H. bispinosa. Recent study has shown that Coxiella-like

Kurilshikov et al., 2015). In this study, PCoA plot based on bacteria are frequently detected in Haemaphysalis ticks, although

unweighted Unifrac distances suggested that tick samples of the not all species carry them (Arthan et al., 2015). Coxiella endosym-

same species, speciﬁcally H. wellingtoni and H. hystricis, shared bionts have also been reported in other tick species such as the R.

similar bacterial communities respectively. However, there was no sanguineus, A. americanum, and the soft tick Ornithodoros rostratus

distinct clustering based on life stages for the H. wellingtoni sam- (Noda et al., 1997; Jasinskas et al., 2007; Almeida et al., 2012). As

ples. High variation was also observed between the H. bispinosa it is beyond the scope of this study, phylogenetic analysis using

samples. When the taxa abundance was taken into account in a greater number of genes in addition to the 16s rRNA sequences

the analysis based on weighted Unifrac distances, differences in was not performed and thus may be required to fully determine

the taxa abundance, and hence the bacterial community structure, the genetic relatedness of the Coxiella strains here compared with

based on tick species and life stages was also undetected. Small the existing strains of Coxiella endosymbionts. The signiﬁcance

sample numbers in this study may pose a limitation in deriving of Coxiella in ticks physiology is still unclear, but there is evi-

biologically meaningful results from diversity analysis, hence larger dence to suggest that endosymbionts can be vertically-transmitted,

sample number may be required for validation. It is also important and contribute to the vitamin and cofactor biosynthesis pathways

to note that only female adult ticks were investigated in this study. (Klyachko et al., 2007; Smith et al., 2015). Although Coxiella was

This current study suggested the presence of Rickettsia in tick detected in all samples here, the high variation in the relative abun-

sources found Malaysia, consistent with previous studies (Kho et al., dance of the endosymbiont raises question on the importance of

2015a). Based on the ampliﬁed partial gltA sequence, R. felis, the the endosymbiont in the physiology of ticks. Previous studies have

etiological agent of ﬂea-borne spotted typhus was detected in shown that Coxiella endosymbionts were not detected in all Haema-

one of our samples. R. felis has been previously identiﬁed in ﬂeas physalis and A. americanum ticks tested (Arthan et al., 2015; Trout

(Ctenocephalides) in Malaysia (Mokhtar and Tay, 2011; Kernif et al., Fryxell and DeBruyn, 2016). These ﬁndings suggested that the pres-

2012) and Thailand (Parola et al., 2003a,b). Although frequently ence of Coxiella endosymbionts might not be absolutely necessary

observed in ﬂeas, molecular detection has also identiﬁed R. felis for the survival of ticks. Therefore, it is possible that not all indi-

in Rhipicephalus sanguineus group ticks in Brazil (Oliveira et al., viduals of the same tick species will harbor the endosymbionts at

2008) and Chile (Abarca et al., 2013), as well as Haemaphysalis similar abundance or some individuals may not carry any Coxiella

ﬂava ticks in Japan (Ishikura et al., 2003), although these studies endosymbionts at all.

did not show evidence of stable infection and disease transmission The speciﬁc etiological agents of tick-borne diseases in Malaysia

by ticks. Nevertheless, human infections of R. felis have been doc- remained to be investigated, and this study suggests that NGS may

umented worldwide, including Thailand and Laos (Parola, 2011), be the tool of choice in screening for these agents. The analysis of

indicating that R. felis is an important emerging pathogen requir- 40,000 sequences per sample was readily sufﬁcient in providing

ing more investigations into the prevalence and its transmission to a picture of the bacterial community proﬁle in our tick samples,

humans or animal hosts in this region. The role of ticks in transmit- suggesting that a larger number of samples (more than 20) could

ting this pathogen needs to be further clariﬁed. Serological assays be sequenced simultaneously in a single run on an Ion Torrent

of human population in the rural areas of peninsula Malaysia or 316 chip. Hence, sequencing on NGS platform may be advanta-

Borneo island demonstrated prevalence of tick-typhus and spot- geous for the purpose of studying a large number of samples. As

ted fever group rickettsiae (Sagin et al., 2000; Tay et al., 2000). amplicon-based sequencing may be biased due to differences in

Our unpublished ﬁndings from serological assays also described primer efﬁciencies or the targeted regions in the 16s rRNA gene

the prevalence of spotted fever among the rural human popula- (Klindworth et al., 2012; Pinto and Raskin, 2012), metagenomic

tion in peninsula Malaysia, however, the exact Rickettsia species sequencing of complete genomic DNA or RNA may be more use-

associated with these cases is still unclear. R. felis may constitute ful for future studies to capture the full microbial diversity. The

a candidate etiological factor that merits further investigation. As need to expand the geographical regions for sampling and increase

we sampled our ticks from domestic animals, which have been the sample size also becomes important now to fully establish the

shown to harbor spotted fever group rickettsiae in earlier studies, geographical presence of these disease agents in Malaysia.

it is also important to determine the possibility of these animals

serving as the reservoir for R. felis (Mokhtar and Tay, 2011; Hii

et al., 2013). Although Rickettsia was detected in 16 samples here, 5. Conclusions

it was observed to be highly abundant in only 3 tick samples. The

impact of bacterial abundance in ticks on the risk of transmission This study presents the assessment of the bacterial communities

is unclear at this point of the study, hence further studies may be associated with Haemaphysalis ticks related to H. wellingtoni, H. hys-

required to correlate the prevalence and abundance of the Rickettsia tricis and H. bispinosa from domestic animals found in the villages

in ticks and the prevalence of rickettsial spotted fever among the of Orang Asli in Malaysia. Diverse array of bacterial communities

human population in the sampling location. Other tick-associated were detected in our tick samples, even though they may comprise

pathogens revealed in this study include Anaplasma, Ehrlichia and of both internal microﬂora of ticks, and the microbial species found

Bartonella, albeit at low abundance in the samples detected. Due on the exoskeleton. We detected potential agents of tick-borne ill-

to the low abundance of these bacteria, the signiﬁcance in disease nesses, speciﬁcally Rickettsia, together with Anaplasma, Ehrlichia

transmission from the ticks in the samplng site is possibly low, too. and Bartonella albeit at low abundance in our samples. The presence

Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013

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TTBDIS-662; No. of Pages 9 ARTICLE IN PRESS

8 J.-J. Khoo et al. / Ticks and Tick-borne Diseases xxx (2016) xxx–xxx

of Rickettsia, especially R. felis, may explain the seroprevalence of sequence formation and detection in Sanger and 454-pyrosequenced PCR

amplicons. Genome Res. 21, 494–504.

spotted fever described in previous serological studies among the

Hamady, M., Lozupone, C., Knight, R., 2010. Fast UniFrac: facilitating

Orang Asli population, although more work is needed to establish

high-throughput phylogenetic analyses of microbial communities including

the link, such as through systematic documentation of tick bites, analysis of pyrosequencing and PhyloChip data. ISME J. 4, 17–27.

Hawlena, H., Rynkiewicz, E., Toh, E., Alfred, A., Durden, L.A., Hastriter, M.W.,

accompanying serological assays and bacterial detection from the

Nelson, D.E., Rong, R., Munro, D., Dong, Q., 2013. The arthropod, but not the

blood samples of patients suspected with tick-borne infections. The

vertebrate host or its environment, dictates bacterial community composition

presence of Coxiella in the Haemaphysalis ticks in Malaysia merits of ﬂeas and ticks. ISME J. 7, 221–223.

Hii, S.-F., Abdad, M.Y., Kopp, S.R., Stenos, J., Rees, R.L., Traub, R.J., 2013.

further investigation as a tick endosymbiont or even as a potential

Seroprevalence and risk factors for Rickettsia felis exposure in dogs from

pathogen. The ﬁndings here provide a baseline knowledge of the

Southeast Queensland and the Northern Territory, Australia. Parasites Vectors

microbiome of ticks in Malaysia. 6, 159.

Hirunkanokpun, S., Kittayapong, P., Cornet, J.-P., Gonzalez, J.-P., 2003. Molecular

evidence for novel tick-associated spotted fever group rickettsiae from

Acknowledgements Thailand. J. Med. Entomol. 40, 230–237.

Irwin, P.J., Jefferies, R., 2004. Arthropod-transmitted diseases of companion

animals in Southeast Asia. Trends Parasitol. 20, 27–34.

This study was supported in parts by the research grants

Ishikura, M., Ando, S., Shinagawa, Y., Matsuura, K., Hasegawa, S., Nakayama, T.,

from the Naval Medical Research Center - Asia and the U.S. Fujita, H., Watanabe, M., 2003. Phylogenetic analysis of spotted fever group

Rickettsiae based on gltA, 17-kDa, and rOmpA genes ampliﬁed by nested PCR

Department of State, Biosecurity Engagement Program (NAMRU:

from ticks in Japan. Microbiol. Immunol. 47, 823–832.

J-55025-75053), University of Malaya, Malaysia (UMRG, RP013-

Jasinskas, A., Zhong, J., Barbour, A.G., 2007. Highly prevalent Coxiella sp bacterium

2012A and RP013-2012B grants), and the Ministry of Higher in the tick vector Amblyomma americanum. Appl. Environ. Microbiol. 73,

334–336.

Education (MOHE) under the High Impact Research (HIR)-MOHE

Kernif, T., Socolovschi, C., Wells, K., Lakim, M.B., Inthalad, S., Slesak, G.,

Grant (E000013-20001) and the Fundamental Research Grant

Boudebouch, N., Beaucournu, J.-C., Newton, P.N., Raoult, D., Parola, P., 2012.

Scheme (FRGS, FP033-2014A). The sequencing were made pos- Bartonella and Rickettsia in arthropods from the Lao PDR and from Borneo,

sible using sequencing infrastructure funded by the Ministry of Malaysia. Comp. Immunol. Microbiol. 35, 51–57.

Kho, K., Koh, F., Tay, S., 2015a. Molecular evidence of potential novel spotted fever

Higher Education, Malaysia Long Term Research (LRGS) Grant

group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks

(LRGS/TD/2011/UM/Penyakit-Berjangkit) and University of Malaya

parasitizing wild snakes. Parasites Vectors 8, 112.

HIR Grant (H-20001-00-E000011). We thank Associate Professor Kho, K.-L., Koh, F.-X., Jaafar, T., Nizam, Q.N.H., Tay, S.-T., 2015b. Prevalence and

molecular heterogeneity of Bartonella bovis in cattle and Haemaphysalis

Dr. Tay Sun Tee and Ms. Hew Shenli for the useful comments on

bispinosa ticks in Peninsular Malaysia. BMC Vet. Res. 11, 1.

the manuscript. We also thank Ms. Chitra Ratnaphongsa, Mr. Khor

Khor, G., Zalilah, M., 2008. The ecology of health and nutrition of Orang Asli

Chee Sieng and Ms. Syuhaida Sulaiman for their technical assistance (Indigenous people) women and children in Peninsular Malaysia. Tribes Tribals

2, 66–77.

in sample preparation and sequencing.

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F.O., 2012. Evaluation of general 16S ribosomal RNA gene PCR primers for

classical and next-generation sequencing-based diversity studies. Nucleic

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Please cite this article in press as: Khoo, J.-J., et al., Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang

Asli communities in Malaysia. Ticks Tick-borne Dis. (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.04.013