Coxiella-like bacteria in fowl ticks from Thailand

Background Coxiella bacteria were identified from various tick species across the world. Q fever is a zoonotic disease caused by the bacteria Coxiella burnetii that most commonly infects a variety of mammals. Non-mammalian hosts, such as birds, have also been reported to be infected with the pathogenic form of “Candidatus Coxiella avium”. This research increases the list of tick species that have been found with Coxiella-like bacteria in Thailand. Methods 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. Ticks were identified and PCR was used to amplify Coxiella bacteria 16S rRNA, groEL and rpoB genes from the extracted tick DNA. MEGA6 was used to construct phylogenetic trees via a Maximum Likelihood method. Results The phylogenetic analysis based on the 16S rRNA gene showed that the Coxiella sequences detected in this study grouped in the same clade with Coxiella sequences from the same tick genus (or species) reported previously. In contrast, rpoB gene of the Coxiella bacteria detected in this study did not cluster together with the same tick genus reported previously. Instead, they clustered by geographical distribution (Thai cluster and Malaysian cluster). In addition, phylogenetic analysis of the groEL gene (the chaperonin family) showed that all Coxiella bacteria found in this study were grouped in the same clade (three sister groups). Conclusions To our knowledge, we found for the first time rpoB genes of Coxiella-like bacteria in Haemaphysalis wellingtoni ticks forming two distinct clades by phylogenetic analysis. This may be indicative of a horizontal gene transfer event.


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.  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].

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].

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 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).
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, Coxiellalike 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.