Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang Asli communities in Malaysia

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Bacterial community in Haemaphysalis ticks of domesticated animals from the Orang Asli communities in Malaysia Jing-Jing Khoo a,b , Fezshin Chen a,b , Kai Ling Kho b , Azzy Iyzati Ahmad Shanizza a,b , Fang-Shiang Lim b , Kim-Kee Tan a,b , Li-Yen Chang a,b , Sazaly AbuBakar a,b,∗ a Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia b 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

Article history: Received 20 January 2016 Received in revised form 8 April 2016 Accepted 20 April 2016 Available online xxx Keywords: Microbiome Coxiella endosymbiont Orang asli Malaysia

a b s t r a c t Ticks are vectors in the transmission of many important infectious diseases in human and animals. Ticks can be readily found in the semi-forested areas such as the settlements of the indigenous people in Malaysia, the Orang Asli. There is still minimal information available on the bacterial agents associated with ticks found in Malaysia. We performed a survey of the bacterial communities associated with ticks 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 hystricis and Haemaphysalis bispinosa. Bacterial 16s rRNA hypervariable region (V6) amplicon libraries prepared from the tick samples were sequenced on the Ion Torrent PGM platform. We detected a total of 392 possible bacterial genera after pooling and sequencing 20 samples, indicating a diverse bacterial community 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 performed 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. © 2016 Published by Elsevier GmbH.

1. Introduction Ticks are excellent vectors for the transmission of zoonotic infectious agents, including bacteria, viruses and protozoan parasites, between human and animals. Ticks are known to harbor a number of medically-important bacterial species within the Rickettsia, Anaplasma, Bartonella, Coxiella and Ehrlichia genera (Petney, 1993; Philippe et al., 2005). As ticks exhibit two-hosts or three-

∗ Corresponding author at: Tropical Infectious Diseases Research and Education Centre (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia. E-mail addresses: [email protected] (J.-J. Khoo), [email protected] (F. Chen), [email protected] (K.L. Kho), azzy [email protected] (A.I. Ahmad Shanizza), [email protected] (F.-S. Lim), [email protected] (K.-K. Tan), [email protected] (L.-Y. Chang), [email protected] (S. AbuBakar).

hosts life cycles, they are capable of supporting the transmission of pathogens between hosts, in which humans frequently serve as the accidental host. Multiple pathogenic agents may also be carried by an individual tick, which could transmit these pathogens to the human hosts during a bite (Carmichael and Fuerst, 2006). Ticks are also known to harbor endosymbionts or commensal bacteria. However, conventional methods for bacteria identification, including molecular or cultivation-dependent techniques, limits the detection 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 unexpected bacteria that may have been missed using conventional methods (Andreotti et al., 2011; Carpi et al., 2011; Menchaca et al., 2013; Nakao et al., 2013; Vayssier-Taussat et al., 2013; WilliamsNewkirk et al., 2014). Tick-borne illness, such as tick typhus and ehrlichiosis, in humans or animals have been documented in Southeast Asian

http://dx.doi.org/10.1016/j.ttbdis.2016.04.013 1877-959X/© 2016 Published by Elsevier GmbH.

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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countries (Petney, 1993; Sirisanthana et al., 1994; Sagin et al., 2000; Tay et al., 2000; Irwin and Jefferies, 2004). Most of the findings were based on serological or microscopic examinations of blood samples from humans or animals, such as domestic dogs or cats, presented with symptoms of tick-borne illnesses. Molecular approaches such as polymerase chain reaction (PCR) have also led to the identification of pathogenic bacteria, including Rickettsia, Ehrlichia and Anaplasma in ticks sampled from forests or villages in Thailand, Philippines, Laos and Malaysia (Hirunkanokpun et al., 2003; Parola et al., 2003a,b; Kernif et al., 2012; Ybanez et al., 2013; Kho et al., 2015a,b). In Malaysia, the indigenous people, also known as the Orang Asli, live in remote villages or settlements within or nearing the tropical rainforests in the Malay peninsula or the Borneo island. The livelihood of most of these communities is still very much dependent on the hunting and foraging activities within the forests (Khor and Zalilah, 2008). The Orang Asli people also live in close contact with domestic animals such as dogs, cats or chickens, which they keep as companion animals or as livestock. As the lifestyle of the Orang Asli involves frequent encounters with wildlife or domestic animals, which are common hosts for ticks, the Orang Asli people are at higher risk of contracting accidental tick bites and tickborne illnesses. There has only been one earlier study investigating the seroprevalence of tick-borne rickettsial infections within the aboriginal communities in Sarawak, Malaysia (Sagin et al., 2000). Pathogen detection in ticks from Malaysia has only been reported in a handful of studies, which identified the presence of spotted fever group rickettsial species and Coxiella from livestock and domestic animals (Tay et al., 2014; Kho et al., 2015a,b; Koh et al., 2015; Watanabe et al., 2015). Hence, more studies are necessary to determine the presence and risks of tick-borne diseases in this region, especially within the communities of the Orang Asli. In this study, we sampled ticks found feeding on domestic animals kept by the Orang Asli from two villages in Malaysia. We investigated the bacterial community harbored by these ticks by using the 16s rRNA V6 hypervariable region amplicon sequencing approach utilizing the Ion Torrent PGM platform technology. 2. Material and methods 2.1. Tick samples Feeding ticks were collected from domestic animals (9 dogs, 6 cats and 5 chickens) observed in 2 separate villages of Orang Asli in the Perak state of Malaysia. All field sampling activities were performed with the approval from the Department of Orangs Asli Development, Malaysia (JAKOA). Once removed from the hosts, the ticks were quick-frozen in liquid nitrogen for transportation and stored in −80 ◦ C until further processing. The collected ticks were microscopically identified to the genus level and classified by life stage according to published taxonomic key for Haemaphysalis ticks (Tanskul and Inlao, 1989). The adult ticks were all identified as female ticks. Adult ticks were processed as individual samples, while nymphs and larvae were pooled into groups of 3–10 depending on the size (Table 1). For DNA extraction, frozen tick samples were thawed, washed thrice in 70% ethanol followed by sterile deionized water to remove environmental debris and disinfect the surface (Carpi et al., 2011). Water washes were subjected to nucleic acid amplification using bacterial-specific primers as described below.

inants and also protection of researchers from potential infectious pathogens. Tick samples were first pulverized in liquid nitrogen using chilled mortar and pestle, using one set for each sample. Before use, all mortars and pestles were soaked in 10% sodium hypochlorite solution for an hour, rinsed with sterile deionized water and baked at 160 ◦ C overnight to eliminate contaminating materials as much as possible. The resulting fine powder was resuspended in 500 ␮L of sterile phosphate-buffered saline (PBS). An aliquot of the suspension (200 ␮L) was used for DNA extraction using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. DNA was eluted in 100 ␮L of Ultrapure DNA/RNA-free distilled water (Invitrogen Life Technologies, MA, USA). Mock extraction was performed in parallel using the same lot of reagents. DNA concentration was determined by spectrophotometry using the Implen Nanophotometer (Implen, Munich, Germany). Molecular identification of the tick samples were performed using previously published primers for the amplification of the mitochondrial 16 rRNA partial sequence (Black and Piesman, 1994). 2.3. Preparation of the barcoded 16s rRNA V6 amplicon libraries The presence of bacteria in the tick samples were detected via nucleic acid amplification using barcode-tagged primers targeting the V6 hypervariable region of the 16s rRNA gene (nt 872–1052 of the complete 16s rRNA sequence of Escherichia coli, NCBI GenBank Accession: AJ605115) as previously described (Carpi et al., 2011). The V6 region allows for most discrimination between pathogenic and non-pathogenic bacteria (Carpi et al., 2011). The forward primers consisted of the A-adaptor sequence (Ion Torrent-specific sequence), an Ion XpressTM barcode sequence (Life Technologies, MA, USA) and a GAT linker fused to the 5 end of the 16s rRNA target sequence. Each of the 20 tick samples was assigned to 1 of the 20 barcode sequences (Ion XpressTM 77–96) used for sample identification and demultiplication during sequence analysis. The reverse primer consisted of a CC linker and the Pi-adaptor sequence (trP1, Ion Torrent-specific sequence) fused to the 5 end of the 16s rRNA target sequence. For the generation of the V6 amplicon library, DNA amplification was performed in 50 ␮L reaction mixture, containing 2 ␮L of extracted DNA, 2.5 units of Dreamtaq DNA polymerase (Thermo Scientific, MA, USA), 0.2 ␮M of dNTPs (Promega, WI, USA), and 0.2 ␮M each of the forward and reverse primers. PCR was performed with the following conditions: activation at 94 ◦ C for 3 min, followed by 32 cycles of denaturation at 94 ◦ C for 1 min, annealing at 56 ◦ C for 1 min and extension at 72 ◦ C for 2 min, with a final elongation step at 72 ◦ C for 2 min. The resulting amplicons were separated by 1.5% agarose gel electrophoresis stained with the SYBR® Safe nucleic acid stain (Invitrogen Life Technologies, MA, USA). All amplicons observed with the expected DNA molecular weight were gel purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. In parallel, negative control PCR reactions were set up using only Ultrapure DNA/RNA-free distilled water (Invitrogen Life Technologies, MA, USA) in place of DNA samples. PCR reactions were also performed with the pre-homogenization water washes and elutions from mock DNA extractions as described above. These reactions did not produce any detectable amplicons upon evaluation with gel electrophoresis and hence excluded from further study. 2.4. Sequencing on the Ion Torrent PGM platform

2.2. DNA extraction from tick samples DNA extraction procedures were performed in a biosafety cabinet to ensure sample protection from environmental contam-

Prior to clonal amplification, concentrations of each amplicon were determined using Qubit dsDNA HS assay kit (Life 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 mitochondrial 16s rRNA sequence (NCBI accession number)

% Identity

BdA1 BdA2 BdA3 BdA4 BdN1 BdN2 BdL1 BcN1 BcL1 BchN1 PdA1 PdA2 PcN1 PcN2 PcL1 PcL2 PchA1 PchN1 PchN2 PchL1

Village B

Dog Dog Dog Dog Dog Dog Dog Cat Cat Chicken Dog Dog Cat Cat Cat Cat Chicken Chicken Chicken Chicken

Adult Adult Adult Adult Nymph Nymph Larva Nymph Larva Nymph Adult Adult Nymph Nymph Larva Larva Adult Nymph Nymph Larva

1 1 1 1 3 5 5 3 10 1 1 1 3 4 5 5 1 1 5 5

H. bispinosa (KC853420.1) H. bispinosa (KC853419.1) H. hystricis (AB819198.1) H. hystricis (AB819198.1) H. bispinosa (KC853419.1) H. wellingtoni (AB819221.1) Not amplifed H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) H. hystricis (AB819197.1) H. hystricis (AB819197.1) H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) H. wellingtoni (AB819221.1) Not amplified H. wellingtoni (AB819221.1)

99% 99% 97% 97% 100% 100%

Village P

100% 100% 100% 100% 99% 100% 100% 97% 99% 100% 100% 100%

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

cons of equal concentration for the same sample were pooled and adjusted to the final concentration of 8–12 pM. Emulsion PCR was carried out with Ion OneTouchTM 200 Template Kit V2 DL (Life Technologies, USA) according to the manufacturer’s protocol PNMAN0007220 Rev. 4.0. The amplified Ion Sphere Particles were enriched using Dynabeads MyOne Streptavidin C1 beads (Life Technologies, MA, USA) and enrichment efficiency was assessed using the Ion Sphere Quality Control Kit (Life Technologies, MA, USA). The sequencing of the amplicons were performed using Ion Sequencing 200 kit (Life Technologies, MA, USA) as previously described (Tan et al., 2015). A total of 20 barcoded samples were pooled and loaded into a single 316 chip (Life Technologies, MA, USA). All resulting sequences were deposited into the European Nucleotide Archive (ENA, Study Accession: PRJEB9707).

2.6. Alpha rarefaction Sequence alignment was performed using the PyNAST (1.2.2) alignment method. The aligned sequences were used to build a phylogenetic tree using FastTree (Price et al., 2009). The resulting tree was used to generate alpha-rarefaction plots, with random subsampling performed at incremental steps of 1000 reads and 10 iterations at each step. 2.7. Beta diversity Beta diversity was calculated using the unweighted and weighted UniFrac metric, followed by Principle Coordinates Analysis (PCoA) using the FastUniFrac workflow (Hamady et al., 2010). 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 Demultiplexed and quality-filtered reads from the Ion Torrent Suite (Version 4.0.2) software were converted into FASTA and quality files using SAMtools (Version 0.1.19, http://samtools.sourceforge.net/). Results were then analyzed using the QIIME workflow (MacQIIME version 1.8.0-20140103) (Caporaso et al., 2010). Firstly, all resulting FASTA files were combined and relabeled to ensure compatibility with the QIIME 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 parameters in QIIME, except to only retain sequences between 125 nt to 220 nt in length. Filtered sequences were subjected to de novo operational taxonomic unit (OTU) picking using the UCLUST method at 97% similarity. Taxonomic assignment was performed using RDP Classifier (Version 2.2) with confidence value of 50% based on the Greengenes reference database (Version 13 8). ChimeraSlayer (Haas et al., 2011) was used to remove chimeric sequences from the OTU Table Singletons were also removed from the OTU table before rarefaction to minimize the inflation of the number of detected OTUs due to potential PCR bias and sequencing error.

PCR amplification of the partial Rickettsia gltA and Coxiella 16s rRNA genes were performed using previously described primers and amplification protocols to detect Rickettsia (Kho et al., 2015a,b) and Coxiella (Duron et al., 2014) respectively. PCR amplicons were electrophoresed and extracted as described above. Sequencing of the amplicons was performed by a third party service provider (First Base Laboratories, Malaysia). The resulting sequences were compared to the available sequences in the NCBI GenBank database (http://www.ncbi.nml.nih.gov/BLAST/). 3. Results 3.1. Tick samples In total, 20 distinct samples were used for sequencing, each consisting of individual adult, female ticks or pools of nymphs or larvae collected from dogs, cats, or chickens as indicated in Table 1. Molecular identification demonstrated 11 samples highly identical (97–100%) to Haemaphysalis wellingtoni (NCBI Accession: AB819221.1), 4 samples similar (97–100%) to Haemaphysalis hystricis (NCBI Accession: AB819198.1), and 3 samples similar (99–100%) 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

Observed species

1600 1400 1200 1000 800 600 400 200 0 0

5000

10000

15000

20000

25000

30000

35000

40000

Number of Reads BdA1

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 PchN2 despite repeated attempts.

In this study, sequencing the amplicons of the V6 hypervariable region of the bacterial 16s rRNA gene on the Ion Torrent PGM platform produced a number of reads ranging from 66118 to 147221 for 19 out of the 20 ticks samples tested (see Table S.1). PchN2 was omitted from further analysis due to its small number of reads (424). Initial quality filtering step in the QIIME workflow yielded 54640–126559 sequences (82.6% to 92.5% of raw sequences) for downstream analysis. To overcome the possible effects of unequal sequencing output across samples, the sequences were rarefied to 40,000 reads for use in OTU picking and taxonomic assignment (Ponnusamy et al., 2014).

(see Table S.1), with nymphs exhibiting higher number of dominant 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 possible clustering of H. wellingtoni and H. hytricis samples respectively, suggesting more similar bacterial community shared by samples of the same tick species (Fig. 3A). H. bispinosa samples showed less defined clustering, with possible overlap with H. wellingtoni and H. hytricis samples. The sole Haemaphysalis sp. sample did not cluster with other tick species. Since the three life stages were only represented in the H. wellingtoni samples, PCoA plot was generated according to life stage for these samples (Fig. 3B). There was no clear clustering of samples based on the different life stages. PCoA plot based on weighted Unifrac distances did not show defined clusterings of samples based on species of life stage (data not shown).

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) were identified for the 19 samples, with 392 taxa assigned to the genus level. Each sample exhibited the number of taxa in the range of 200–334 (see Table S.1). The rarefaction curves each of the tick samples approached saturation at 40,000 reads (Fig. 1), indicating sufficient sampling depth. At the class level, taxonomical composition of the 19 tick samples indicated that the dominant bacterial classes in the samples are Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, Bacilli and Deltaproteobacteria, in combination representing 80% to 99% of the population in each of the sample (Fig. 2). Only 10 bacterial taxa were most represented (≥1%) for the 19 samples combined (Table 2), in which 8 out of 10 bacterial taxa were assigned to the genus level. Collectively, 0.97% of the reads in the 19 samples were unassigned to any taxon beyond the bacteria domain. In each sample, the number of dominant taxa (≥1%) ranged from 2 to 24 taxa

A number of bacterial genera commonly associated with ticks were observed in these samples. Coxiella accounted for the most abundant bacterial taxa (39.2%) in all samples collectively (Table 2). Coxiella was detected in all 19 samples, ranging from 0.23% to 89.38% in each sample (Table 3), with a number of nymphs and larvae samples exhibiting lower relative abundance (BdL1 and BcN1). Rickettsia was the second most abundant bacterial taxa collectively (6%, Table 2). Rickettsia was observed in 16 out of 19 samples, ranging from 0.01% to 55.1% of the total reads in each sample (Table 3). Specifically, Rickettsia was highly abundant in 3 samples, namely BdN2 (37.12%), BdL1 (55.1%), and PcL2 (15.95%). Anaplasma and Ehrlichia were detected in 3 out of 19 samples, albeit at low abundance (less than 1%, Table 3). BcN1 exhibited the highest level of Anaplasma (0.18%) and Ehrlichia (0.16%) out of the 3 samples detected. Although not assigned to the genus level, PcN1 displayed approximately 10% reads assigned to Anaplasmataceae

3.2. 16s rRNA V6 amplicon sequencing results

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 k k k k k k k k k k

Bacteria;p Bacteria;p Bacteria;p Bacteria;p Bacteria;p Bacteria;p Bacteria;p Bacteria;p Bacteria;p Bacteria;p

Abundance (%) Proteobacteria;c Gammaproteobacteria;o Legionellales; f Coxiellaceae;g Coxiella Proteobacteria;c Alphaproteobacteria;o Rickettsiales; f Rickettsiaceae;g Rickettsia Proteobacteria;c Alphaproteobacteria;o Rhizobiales; f Phyllobacteriaceae;g Firmicutes;c Bacilli;o Bacillales;f Bacillaceae; g Bacillus Proteobacteria;c Gammaproteobacteria;Other;Other;Other Actinobacteria;c Actinobacteria;o Actinomycetales; f Mycobacteriaceae;g Mycobacterium Proteobacteria;c Alphaproteobacteria;o Sphingomonadales;f Sphingomonadaceae;g Sphingomonas Proteobacteria;c Gammaproteobacteria;o Pseudomonadale;f Pseudomonadaceae;g Pseudomonas Proteobacteria;c Alphaproteobacteria;o Rhizobiales; f Methylobacteriaceae;g Methylobacterium Firmicutes;c Bacilli;o Bacillales;f Staphylococcaceae; g Staphylococcus

39.2 6.0 4.7 3.8 2.9 2.8 1.7 1.5 1.3 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 potentially similar to the other members of Anaplasmataceae. Bartonella, another group of tick-associated pathogen, was detected at very low abundance (0.003% to 0.03%) in 5 samples (Table 3). These bacteria were detected together with the presence of Rickettsia in the same samples, indicating possible coinfection. Other well studied tick-associated pathogens, Borrelia and Francisella, were not detected in these samples. Wolbachia and Rickettsiella, which are possible endosymbionts in ticks apart from Coxiella, were also detected in these tick samples. Rickettsiella was observed in 16 samples at low abundance (0.01% to 0.58% in each sample). Wolbachia was highly represented (8.4%) in 1 sample, BdA4, while being detectable at low abundance (0.01% to 0.18%) in additional 5 samples. There was also numerous bacterial genera identified in our tick samples, which consists of species that are potentially pathogenic

A

to humans or animals, but have limited information on the role of ticks in disease transmission. Mycobacterium, Pseudomonas and Staphylococcus were amongst the 10 most abundant taxa in the 19 samples collectively, at 2.8%, 1.5% and 1.3% respectively (Table 2). These bacterial taxa were observed in all samples (Table 3). Examples of other less abundant bacterial taxa detected include Acinetobacter (19/19, positive/total samples), Corynebacterium (19/19), Streptococcus (18/19), Stenotrophomonas (18/19), Clostridium (13/19), Klebsiella (8/19), Serratia (3/19) and Leptospira (3/19) (see Table S.2). Bacteria normally associated with the environment or the soil were also detected in our samples. Specifically, Bacillus, Sphingomonas and Methylobacterium, which were previously reported to be detected in ticks, were detected in all samples studied (Tables 2 and 3).

B

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

19 19 19 0.50 1.49 0.82 0.50 5.33 1.30 0.05 0.18 0.24 0.06 0.42 0.19 0.03 0.28 0.29 0.66 2.31 5.22 0.03 1.44 2.56 0.31 0.08 0.03 1.64 6.55 1.00 6.54 0.97 0.29

35.12 0.58 0.16

5.93 2.34 0.70

0.72 1.43 2.17

0.43 0.91 1.11

0.44 0.29 0.05

0.82 5.93 6.56

1.66 0.42 0.19 Environmental bacteria 16.36 Bacillus 1.60 Sphingomonas 2.26 Methylobacterium

0.13 0.19 0.05

19 19 19 7.96 2.08 0.16 1.33 2.80 3.74 0.26 0.07 0.44 0.39 0.28 0.07 0.30 2.98 0.21 2.98 1.83 1.25 5.48 1.01 0.43 0.15 0.06 0.04 0.09 0.48 0.82 0.89 1.08 4.40 Abundant taxa with potentially pathogenic members but unknown transmission status in ticks 15.36 0.47 0.94 1.24 0.62 1.84 1.67 9.49 1.41 Mycobacterium 0.55 0.13 0.12 1.73 5.18 2.46 3.08 1.24 2.00 Pseudomonas 3.97 1.58 0.07 0.45 1.22 1.11 1.32 0.43 2.26 Staphylococcus

19 16 6 58.55 0.08 0 8.84 0.01 0 89.38 0.01 0.07 73.70 0.02 0.03 61.07 0.03 0 12.29 0.58 0.18 47.43 0.16 0 86.16 0 0.01 78.99 0.04 0 16.36 0.17 0 61.20 0.01 0 0.23 0.13 0 0.26 0.05 0 4.64 0.02 0 8.52 0.01 0.06 27.07 0.03 8.4 29.82 0.14 0

16 3 3 5 0 0 0 0 0.18 0 0 0 0.03 0 0 0 15.95 0.005 0 0.003 0.01 0 0 0 0.47 0 0 0 0.07 0 0 0 0.88 0 0 0 0 0 0.02 0 0.01 0 0 0 0.64 0 0 0 0.10 0.18 0.16 0.03 55.10 0 0 0 37.12 0 0 0 0.48 0 0 0.003 0.86 0.003 0.003 0.02

PcN2 PcN1 PdA2 PdA1 BchN1 BcL1 BcN1 BdL1 BdN2 BdN1 BdA4 BdA3 BdA2 BdA1

Relative abundance (%) Samples

Table 3 Relative abundance of selected bacterial taxa in each tick sample.

In this study, we present a survey of the bacterial community in Haemaphysalis ticks in Malaysia obtained using the NGS technology. These ticks were sampled from the domestic animals (dogs, cats and chickens) observed in villages of the local indigenous people, the Orang Asli, from two different locations in Malaysia. Haemaphysalis ticks are the dominant ticks in our sampled sites, consisting of species identical or related to H. wellingtoni, H. hystricis and H. bispinosa. This observation is consistent with previous reports suggesting the abundance of Haemaphysalis ticks in Malaysia (Audy et al., 1960; Paramasvaran et al., 2009) and also the prevalence of these ticks in domestic animals in South East Asia (Irwin and Jefferies, 2004). Ticks of the Haemaphysalis genus had been implicated as potential disease vector to humans and animals worldwide. Various pathogenic bacteria have been previously detected in Haemaphysalis ticks including the disease agents for rickettsial spotted fever, tick typhus, anaplasmosis and ehrlichiosis (Hirunkanokpun et al., 2003; Liu et al., 2013; Tijsse-Klasen et al., 2013; Yu et al., 2015). In total, 392 bacterial taxa were identified to genus level in our study. Although not directly comparable, metagenomics sequencing in previous reports, which sequenced full genomic DNA, yielded 138 and 156 genera respectively in Haemaphysalis formosensis and H. longicornis (Nakao et al., 2013). Microbiome studies performed on other tick species revealed a variation in numbers of bacterial genus identified. Carpi et al. (2011), who targeted the same V6 region of the 16s rRNA gene, detected 108 bacterial genera in Ixodes ricinus. The cattle tick, Rhipicephalus microplus, was found to harbor 121 bacterial genera (Andreotti et al., 2011). In Amblyomma americanum, two separate studies found 237 (Ponnusamy et al., 2014) and 99 (Williams-Newkirk et al., 2014) genera, respectively. The vast difference in the numbers of genera may be due to the variations in the sequencing platform technology or analytical procedures employed. In addition, the high number of bacterial genera may include bacterial species that were found on the surface of the tick samples, which may have originated from the environment such as the soil or the skin surface of the host mammal despite our efforts in washing the sample. Previous studies have demonstrated a substantial difference in bacterial community composition between surface-sterilized (DNA removed with sodium hypochlorite) and non-sterilized A. americanum tick samples (Menchaca et al., 2013). Since bacterial taxa such as Bacillus, Clostridium, Methylobacterium, Mycobacterium, Pseudomonas, Sphingomonas and Staphylococcus, some of which were found in high abundance in our samples, comprise of species that may be

PcL1

4. Discussion

1.28 0 0 0.01

PcL2

PchA1

PchN1

PchL1

Out of the 3 samples with the highest abundance of Rickettsia (BdN2, BdL1, and PcL2), we successfully amplified and sequenced the partial gltA gene from BdL1 (NCBI Genbank Accession: KU948227), in which the sequence was 100% identical to Rickettsia felis (NCBI Accession: JN375500.1). Sequencing of the gltA gene fragment from PcL2 generated less than satisfactory results despite multiple attempts. No amplification was observed for BdN2. Hence, the Rickettsia strain in PcL2 and BdN2 could not be confirmed in this study. The partial 16s rRNA sequences for Coxiella were successfully amplified from 5 (ENA Accession: LT548048–LT548052) out of all 19 samples tested. The Coxiella strains in BdN2 (96%), BcL1 (96%), PdA1 (97%) and PcL1 (96%) were closely similar to the Haemaphysalis longicornis Coxiella symbiont (NCBI Accession: AY342035.1). The Coxiella strain in BdA2 was highly similar (96%) to the Coxiella symbiont in Haemaphysalis shimoga (NCBI Accession: HQ287535.1).

Medically-important bacteria in ticks 0.08 0 Rickettsia 0 0 Anaplasma 0 0 Ehrlichia 0 0 Bartonella

3.5. PCR detection of Rickettsia and Coxiella

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16.19 0 0

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commonly associated with the environment or as part of the mammalian skin flora, it is possible that these bacteria orginated from the external surfaces of the ticks in our study. More stringent washing procedure, such as washing with sodium hypochlorite, or limiting sampling to internal organs such as the salivary glands and mid-gut, may be necessary to determine the internal flora of ticks in further studies. A number of studies have reported distinct bacterial community structure based on tick species and life stage for Dermacentor variabilis, Dermacentor reticulatus, Ixodes scapularis, Ixodes persulcatus and Ixodes pavlovskyi respectively (Hawlena et al., 2013; Kurilshikov et al., 2015). In this study, PCoA plot based on unweighted Unifrac distances suggested that tick samples of the same species, specifically H. wellingtoni and H. hystricis, shared similar bacterial communities respectively. However, there was no distinct clustering based on life stages for the H. wellingtoni samples. High variation was also observed between the H. bispinosa samples. When the taxa abundance was taken into account in the analysis based on weighted Unifrac distances, differences in the taxa abundance, and hence the bacterial community structure, based on tick species and life stages was also undetected. Small sample numbers in this study may pose a limitation in deriving biologically meaningful results from diversity analysis, hence larger sample number may be required for validation. It is also important to note that only female adult ticks were investigated in this study. This current study suggested the presence of Rickettsia in tick sources found Malaysia, consistent with previous studies (Kho et al., 2015a). Based on the amplified partial gltA sequence, R. felis, the etiological agent of flea-borne spotted typhus was detected in one of our samples. R. felis has been previously identified in fleas (Ctenocephalides) in Malaysia (Mokhtar and Tay, 2011; Kernif et al., 2012) and Thailand (Parola et al., 2003a,b). Although frequently observed in fleas, molecular detection has also identified R. felis in Rhipicephalus sanguineus group ticks in Brazil (Oliveira et al., 2008) and Chile (Abarca et al., 2013), as well as Haemaphysalis flava ticks in Japan (Ishikura et al., 2003), although these studies did not show evidence of stable infection and disease transmission by ticks. Nevertheless, human infections of R. felis have been documented worldwide, including Thailand and Laos (Parola, 2011), indicating that R. felis is an important emerging pathogen requiring more investigations into the prevalence and its transmission to humans or animal hosts in this region. The role of ticks in transmitting this pathogen needs to be further clarified. Serological assays of human population in the rural areas of peninsula Malaysia or Borneo island demonstrated prevalence of tick-typhus and spotted fever group rickettsiae (Sagin et al., 2000; Tay et al., 2000). Our unpublished findings from serological assays also described the prevalence of spotted fever among the rural human population in peninsula Malaysia, however, the exact Rickettsia species associated with these cases is still unclear. R. felis may constitute a candidate etiological factor that merits further investigation. As we sampled our ticks from domestic animals, which have been shown to harbor spotted fever group rickettsiae in earlier studies, 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, it was observed to be highly abundant in only 3 tick samples. The impact of bacterial abundance in ticks on the risk of transmission is unclear at this point of the study, hence further studies may be required to correlate the prevalence and abundance of the Rickettsia in ticks and the prevalence of rickettsial spotted fever among the human population in the sampling location. Other tick-associated pathogens revealed in this study include Anaplasma, Ehrlichia and Bartonella, albeit at low abundance in the samples detected. Due to the low abundance of these bacteria, the significance in disease transmission from the ticks in the samplng site is possibly low, too.

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We observed Coxiella in our samples, which was also detected in a previous study of Haemapysalis ticks (Nakao et al., 2013). Due to the short sequence of the 16s rRNA V6 region used in NGS, we were unable to differentiate between Coxiella endosymbionts or Coxiella burnetii. However, PCR detection based on longer but partial 16s rRNA sequences amplified from 5 samples indicated the presence of Coxiella symbionts closely similar to previously identified Coxiella endosymbionts in H. longicornis (Lee et al., 2004) and H. shimoga (Ahantarig et al., 2011). Our study reports the presence of Coxiella 16s rRNA partial sequence in ticks related to H. wellingtoni, H. hystricis or H. bispinosa. Recent study has shown that Coxiella-like bacteria are frequently detected in Haemaphysalis ticks, although not all species carry them (Arthan et al., 2015). Coxiella endosymbionts have also been reported in other tick species such as the R. sanguineus, A. americanum, and the soft tick Ornithodoros rostratus (Noda et al., 1997; Jasinskas et al., 2007; Almeida et al., 2012). As it is beyond the scope of this study, phylogenetic analysis using a greater number of genes in addition to the 16s rRNA sequences was not performed and thus may be required to fully determine the genetic relatedness of the Coxiella strains here compared with the existing strains of Coxiella endosymbionts. The significance of Coxiella in ticks physiology is still unclear, but there is evidence to suggest that endosymbionts can be vertically-transmitted, and contribute to the vitamin and cofactor biosynthesis pathways (Klyachko et al., 2007; Smith et al., 2015). Although Coxiella was detected in all samples here, the high variation in the relative abundance of the endosymbiont raises question on the importance of the endosymbiont in the physiology of ticks. Previous studies have shown that Coxiella endosymbionts were not detected in all Haemaphysalis and A. americanum ticks tested (Arthan et al., 2015; Trout Fryxell and DeBruyn, 2016). These findings suggested that the presence of Coxiella endosymbionts might not be absolutely necessary for the survival of ticks. Therefore, it is possible that not all individuals of the same tick species will harbor the endosymbionts at similar abundance or some individuals may not carry any Coxiella endosymbionts at all. The specific etiological agents of tick-borne diseases in Malaysia remained to be investigated, and this study suggests that NGS may be the tool of choice in screening for these agents. The analysis of 40,000 sequences per sample was readily sufficient in providing a picture of the bacterial community profile in our tick samples, suggesting that a larger number of samples (more than 20) could be sequenced simultaneously in a single run on an Ion Torrent 316 chip. Hence, sequencing on NGS platform may be advantageous for the purpose of studying a large number of samples. As amplicon-based sequencing may be biased due to differences in primer efficiencies or the targeted regions in the 16s rRNA gene (Klindworth et al., 2012; Pinto and Raskin, 2012), metagenomic sequencing of complete genomic DNA or RNA may be more useful for future studies to capture the full microbial diversity. The need to expand the geographical regions for sampling and increase the sample size also becomes important now to fully establish the geographical presence of these disease agents in Malaysia.

5. Conclusions This study presents the assessment of the bacterial communities associated with Haemaphysalis ticks related to H. wellingtoni, H. hystricis and H. bispinosa from domestic animals found in the villages of Orang Asli in Malaysia. Diverse array of bacterial communities were detected in our tick samples, even though they may comprise of both internal microflora of ticks, and the microbial species found on the exoskeleton. We detected potential agents of tick-borne illnesses, specifically Rickettsia, together with Anaplasma, Ehrlichia 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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of Rickettsia, especially R. felis, may explain the seroprevalence of spotted fever described in previous serological studies among the Orang Asli population, although more work is needed to establish the link, such as through systematic documentation of tick bites, accompanying serological assays and bacterial detection from the blood samples of patients suspected with tick-borne infections. The presence of Coxiella in the Haemaphysalis ticks in Malaysia merits further investigation as a tick endosymbiont or even as a potential pathogen. The findings here provide a baseline knowledge of the microbiome of ticks in Malaysia. Acknowledgements This study was supported in parts by the research grants from the Naval Medical Research Center - Asia and the U.S. Department of State, Biosecurity Engagement Program (NAMRU: J-55025-75053), University of Malaya, Malaysia (UMRG, RP0132012A and RP013-2012B grants), and the Ministry of Higher Education (MOHE) under the High Impact Research (HIR)-MOHE Grant (E000013-20001) and the Fundamental Research Grant Scheme (FRGS, FP033-2014A). The sequencing were made possible using sequencing infrastructure funded by the Ministry of Higher Education, Malaysia Long Term Research (LRGS) Grant (LRGS/TD/2011/UM/Penyakit-Berjangkit) and University of Malaya HIR Grant (H-20001-00-E000011). We thank Associate Professor Dr. Tay Sun Tee and Ms. Hew Shenli for the useful comments on the manuscript. We also thank Ms. Chitra Ratnaphongsa, Mr. Khor Chee Sieng and Ms. Syuhaida Sulaiman for their technical assistance in sample preparation and sequencing. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ttbdis.2016.04. 013. References Abarca, K., López, J., Acosta-Jamett, G., Martínez-Valdebenito, C., 2013. Rickettsia felis in Rhipicephalus sanguineus from two distant Chilean cities. Vector Borne Zoonotic Dis. 13, 607–609. Ahantarig, A., Malaisri, P., Hirunkanokpun, S., Sumrandee, C., Trinachartvanit, W., Baimai, V., 2011. Detection of Rickettsia and a novel Haemaphysalis shimoga symbiont bacterium in ticks in Thailand. Curr. Microbiol. 62, 1496–1502. Almeida, A.P., Marcili, A., Leite, R.C., Nieri-Bastos, F.A., Domingues, L.N., Martins, J.R., Labruna, M.B., 2012. Coxiella symbiont in the tick Ornithodoros rostratus (Acari: argasidae). Ticks Tick Borne Dis. 3, 203–206. Andreotti, R., Perez de Leon, A., Dowd, S., Guerrero, F., Bendele, K., Scoles, G., 2011. Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing. BMC Microbiol. 11, 6. Arthan, W., Sumrandee, C., Hirunkanokpun, S., Kitthawee, S., Baimai, V., Trinachartvanit, W., Ahantarig, A., 2015. Detection of Coxiella-like endosymbiont in Haemaphysalis tick in Thailand. Ticks Tick Borne Dis. 6, 63–68. Audy, J.R., Nadchatram, M., Lim, B.-L., 1960. Malaysian parasites XLIX. host distribution of malayan ticks (Ixodoidea). Stud. Inst. Med. Res. Malaya, 225–246. Black, W.C., Piesman, J., 1994. Phylogeny of hard- and soft-tick taxa (Acari: ixodida) based on mitochondrial 16S rDNA sequences. Proc. Natl. Acad. Sci. U. S. A. 91, 10034–10038. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. Carmichael, J.R., Fuerst, P.A., 2006. A Rickettsial mixed infection in a Dermacentor Variabilis tick from Ohio. Ann. N. Y. Acad. Sci. 1078, 334–337. Carpi, G., Cagnacci, F., Wittekindt, N.E., Zhao, F., Qi, J., Tomsho, L.P., Drautz, D.I., Rizzoli, A., Schuster, S.C., 2011. Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks. PLoS One 6, e25604. Duron, O., Jourdain, E., McCoy, K.D., 2014. Diversity and global distribution of the Coxiella intracellular bacterium in seabird ticks. Ticks Tick Borne Dis. 5, 557–563. Haas, B.J., Gevers, D., Earl, A.M., Feldgarden, M., Ward, D.V., Giannoukos, G., Ciulla, D., Tabbaa, D., Highlander, S.K., Sodergren, E., 2011. Chimeric 16S rRNA

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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