Background

Ticks are ectoparasites of vertebrates transmitting pathogens like protozoa, viruses and bacteria which cause zoonotic diseases in domestic animals and humans. Both hard and soft tick species have been documented to harbour Coxiella bacteria. For example, the soft ticks Ornithodoros capensis (s.l.), O. rostratus and the hard ticks Dermacentor atrosignatus and Amblyomma testudinarium have been found to be infected with Coxiella bacteria [1,2,3,4].

Q fever (Query fever) is a zoonotic disease caused by Coxiella burnetii that most commonly infects a variety of mammals throughout the world. Coxiella burnetii affects the reproductive system in animals causing stillbirths and miscarriages. Infection of C. burnetii in humans results from inhalation of contaminated aerosols in nature or from direct contact with infected domestic animals products or formites [5, 6]. Individuals infected with C. burnetii were detected among rice farmers who raised cattle and chickens in northeastern Thailand. A seroepidemiological survey of C. burnetii in cattle and chickens in Thailand was carried out using an indirect fluorescent antibody test. Only one out of 113 serum samples from fowl was seropositive [7]. Moreover, non-mammalian hosts, such as birds, have also been reported to be infected with “Candidatus Coxiella avium” [8].

Coxiella bacteria were identified from various species of Haemaphysalis and Rhipicephalus sanguineus (s.l.) ticks in Thailand [3, 9]. Nevertheless, in Thailand, infection of Coxiella bacteria in fowl ticks has rarely been investigated. Therefore, the aims of this work were to determine the presence of Coxiella bacteria in fowl ticks and to study their evolutionary relationships in phylogenetic analyses based on partial 16S, rpoB (RNA polymerase beta-subunit), and groEL (the chaperonin family) gene sequences.

Methods

Tick samples and identification

A total of 69 ticks were collected from 27 domestic fowl (Gallus gallus domesticus), 2 jungle fowl (Gallus gallus) and 3 Siamese firebacks (Lophura diardi) at 10 locations (provinces) in Thailand during 2014–2016: (i) Chaiyaphum; (ii) Chumphon; (iii) Krabi; (iv) Pattani; (v) Rayong; (vi) Satun; (vii) Songkhla; (viii) Surat Thani; (ix) Trang; and (x) Yala (Table 1). The ticks were removed by using forceps, stored in 70% alcohol and preserved at -20 °C awaiting further identification and molecular assays. Ticks were classified by developmental stage and sex and identified based on morphology using standard identification keys [10, 11]. Ticks positive for Coxiella bacteria were also molecularly identified using a primer set consisting of 16S + 1 and 16S - 1 to detect tick 16S mitochondrial DNA (16S mDNA) [12].

Table 1 Tick samples from domestic fowl (Gallus gallus domesticus), jungle fowl (Gallus gallus) and Siamese fireback (Lophura diardi)
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DNA extraction from tick samples and PCR

Ticks were washed with 70% ethanol and 10% sodium hypochlorite and rinsed three times with sterile distilled water. Then, ticks were immediately homogenized using the TissueLyser system (QiagenGmbH, Hilden , Germany). One 3-mm tungsten carbide bead (Qiagen GmbH, Germany) was added to each tube (collection microtubes; Qiagen GmbH, Germany) and ticks (individual for adult, individual for Amblyomma nymph and a pool of 5 for nymph of Haemaphysalis) were homogenized for 4 min at 30 Hz. After a short centrifugation step (5 s at 3220× g), the supernatants were collected in separate collection microtubes and DNA extracted using Qiagen’s DNeasy Blood and Tissues Kit (Qiagen GmbH, Germany) following the manufacturer’s instructions. Genes and primers used to amplify Coxiella DNA were used as in a previously reported protocol [13].

Purification and sequencing of PCR products

After PCR amplification and gel electrophoresis, DNA bands corresponding to positive amplification results were excised. Purified DNA samples (using Purification kit from Roche, Basel, Switzerland) were sent to the Ramathibodi Research Department (Ramathibodi Hospital, Bangkok, Thailand) for DNA sequencing. The results were analysed and compared with other DNA sequences from GenBank in the National Center for Biotechnology Information database (NCBI: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch).

Phylogenetic analyses

DNA sequences were edited and aligned with MEGA6 using ClustalW multiple sequence alignment algorithm. DNA sequences from this study, along with selected reference strains from GenBank, were used to construct a phylogenetic tree via Maximum Likelihood (Kimura 2-parameter model) and determining the confidence value for each branch of the phylogenetic tree with bootstrap analysis by using 1000 pseudoreplicates of the original alignment.

Results and Discussion

A total of 69 ticks collected from domestic and jungle fowl and Siamese firebacks belong to 3 genera: Haemaphysalis; Amblyomma; and Rhipicephalus (Table 1). The 51 adult ticks included 14 R. microplus, 37 H. wellingtoni and the remaining 18 ticks were nymphs of H. wellingtoni (n = 7), H. obesa (n = 5), H. bispinosa (n = 3) and A. testudinarium (n = 3). The 16S mDNA sequences of ticks were submitted to the GenBank database under the accession numbers MG865746 (H. wellingtoni), MG874025, MG910463 and MG874022 (H. obesa, H. bispinosa, and A. testudinarium, respectively). A total of 14 out of 69 ticks tested were positive for Coxiella bacteria, as defined by the amplification of 16S rRNA sequences. Positive results were found in H. wellingtoni from Chumphon, Krabi, Rayong, Satun and Trang, H. obesa from Chaiyaphum, H. bispinosa from Krabi and A. testudinarium from Chaiyaphum (Table 1).

Coxiella bacteria-positive samples were sequenced, and a phylogenetic tree was constructed based on their analysis (Fig. 1). Coxiella DNA sequences were submitted to the GenBank database, including H. wellingtoni from Trang (TRG32 and TRG33), H. obesa and A. testudinarium from Chaiyaphum (PK179-183 and PK190), H. wellingtoni from Rayong (RYG1), H. wellingtoni from Satun (STN77) and H. bispinosa from Krabi (KBI32) (see Fig. 1 for accession numbers).

Fig. 1
figure 1

Phylogenetic tree for Coxiella-like bacteria 16S rRNA gene sequences constructed with the Maximum Likelihood method using MEGA6 software. Bootstrap analysis was performed with 1000 pseudoreplicates. Rickettsia rickettsii was used as the outgroup. Coxiella-like bacteria isolates from this study are indicated in bold and with asterisks

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The phylogenetic analysis, based on the 16S rRNA gene, showed that Coxiella bacteria from Haemaphysalis ticks of domestic fowl and Siamese firebacks were grouped with Coxiella bacteria of the same corresponding tick species previously reported (Fig. 1). In addition, the 16S rRNA sequence of A. testudinarium of our study was in the same group with those reported by Nooroong et al. [3] and Khoo et al. [14] (Fig. 1).

Phylogenetic analyses of rpoB and groEL genes of Coxiella-like bacteria were also performed. The results are shown in Figs. 2 and 3. Most of Coxiella rpoB sequences from this study were in the same group and exhibited 88–89% identity with Coxiella-like endosymbiont of Argas reflexus (isolate Areflex2, GenBank: KY677983) and Coxiella endosymbiont of ticks of the genus Ixodes (GenBank: KP985313, KP985318 and KP985320) (Fig. 2). However, rpoB gene sequences of Coxiella-like bacteria in H. wellingtoni from Trang (TRG33) clustered in a different clade and was closely related to Coxiella endosymbiont of Rhipicephalus sp. isolate (Tchien14; GenBank: KP985345; 96% identity) (Fig. 2). The rpoB gene sequences of Coxiella bacteria detected in this study did not cluster together with those previously reported in the same tick species by Khoo et el. [14] (H. bispinosa and Amblyomma spp.). Instead they seemed to be clustering by their geographical distribution forming a Thai cluster (the present study) and a Malaysian cluster (data by Khoo et al. [14]). Coxiella groEL gene sequences detected in ticks from domestic fowl and Siamese firebacks from this study were clustered in the same clade (three sister groups) and exhibited about 89% DNA sequence identity with “Candidatus Coxiella avium” from seabird ticks (GenBank: KJ459059) (Fig. 3).

Fig. 2
figure 2

Phylogenetic tree for Coxiella-like bacteria rpoB gene sequences constructed with the Maximum Likelihood method using MEGA6 software. Bootstrap analysis was performed with 1000 pseudoreplicates. Rickettsia raoultii was used as the outgroup. Coxiella-like bacteria isolates from this study are indicated in bold and with asterisks

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Fig. 3
figure 3

Phylogenetic tree for Coxiella-like bacteria groEL gene sequences constructed with the Maximum Likelihood method using MEGA6 software. Bootstrap analysis was performed with 1000 pseudoreplicates. Rickettsia tipulae was used as the outgroup. Coxiella-like bacteria isolates from this study are indicated in bold and with asterisks

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Coxiella bacteria have been reported in several tick species, such as Rhipicephalus sanguineus (s.l.), Amblyomma americanum, Ixodes uriae and the soft tick O. rostratus [1, 2, 15, 16]. In addition, Coxiella-like bacteria were also detected in Haemaphysalis ticks, such as H. lagrangei, H. obesa, H. shimoga and H. hystricis [3]. In the present study, the rate of Coxiella-like bacteria in ticks collected from fowl was rather high because about 20% of ticks of the 4 species (H. wellingtoni, H. bispinosa, H. obesa and A. testudinarium) were positive for Coxiella-like bacteria. Thus, our results seem to agree with those of Arthan et al. [4] who demonstrated that the prevalence was not dependent on tick species.

Analyses of rpoB sequences revealed that most of Coxiella-like bacteria exhibited 88–89% identity with Coxiella-like endosymbiont from Argas reflexus (isolate Areflex2). However, rpoB gene of Coxiella bacteria in H. wellingtoni from Trang (TRG33) was clustered in the different group in the phylogenetic analysis and related to Coxiella-like bacteria of Rhipicephalus sp. isolate Tchien14 rpoB gene, partial cds (96% identity). This result may simply be indicative of a horizontal gene transfer event. Since only a small number of sequences is reported here, and only one with these characteristics is shown, it remains to be determined what is the real impact of this observation. The interesting point is that Coxiella rpoB sequences from different H. wellingtoni ticks belong to a different clade (even from the same tick species). The roles of these Coxiella-like bacteria in ticks and their fowl hosts are still unclear and needs further investigation.

Conclusions

To our knowledge, we found for the first time that Coxiella rpoB gene sequences from different H. wellingtoni ticks belong to two different clades and that rpoB sequence of the Coxiella bacteria detected in this study did not cluster together with those previously reported in the same tick species.