Tick-borne agents in the fowl tick Argas persicus from northwest and northeast China

Abstract

Although tick-borne agents have been extensively studied, etiological investigations on soft ticks are still relatively rare. In this study, we collected 114 Argas persicus ticks from two provinces (Xinjiang and Heilongjiang) located in northwest and northeast China, respectively, and screened them for tick-borne agents. Two Rickettsia species were identified in A. persicus ticks from Heilongjiang Province: Rickettsia hoogstraalii (27.3%, 18/66) and a previously unidentified species (12.2%, 8/66). The 16S rDNA, gltA, groEL, and ompB genes of the latter have 98.8%, 93.1%, 94.3%, and 91.2% nucleotide identities to reported species, suggesting that it represents a novel species. It belongs to the ancient group of Rickettsia and is located in the basal position of the phylogenetic trees. Additionally, Coxiella endosymbiont was detected in A. persicus ticks from both locations with 100% positive rates. Furthermore, the Coxiella endosymbionts from different locations form distinct phylogenetic groups, indicating that one tick species can harbor different Coxiella endosymbionts.

Graphical abstract

The fowl tick Argas persicus (Ixodida: Argasidae) is a soft tick commonly associated with poultry [1]. Although its major natural hosts are pigeons, swallows, and chickens, it is sometimes found on domestic animals such as sheep and cattle [2]. It is also reported to attack humans (especially in early Persia), making it possible to transmit pathogens from animals to humans [3]. Tick-borne agents (e.g., Rickettsia, Anaplasma, Coxiella, Borrelia, Babesia, Crimean-Congo hemorrhagic fever virus, and thrombocytopenia syndrome virus) have been extensively studied in China [4]. However, most investigations only focus on hard ticks (family Ixodidae), probably because of their easy availability. Although soft ticks are widely spread in China, their associated agents have been rarely reported. Herein, we collected A. persicus ticks in northwest and northeast China and studied the tick-borne agents in them.

From 2012 to 2017, soft ticks were collected in two locations: the 170 Regiment of the 9th Division in Xinjiang Uygur Autonomous Region, northwest China, and Hegang City in Heilongjiang Province, northeast China. A total of 48 ticks were collected in chicken coops in Xinjiang in the spring of 2012, and 66 ticks were collected in cattle sheds in Heilongjiang in the spring of 2017. The ticks were carefully collected using tweezers. Using taxonomic keys [5], all the ticks were morphologically identified as adult A. persicus (Additional file 1: Figure S1). The genomic DNA of the ticks was extracted using the EZNA® Mollusc & Insect DNA Kit (Omega) according to the manufacturer's instructions. The tick species was confirmed by amplifying and analyzing a 674-bp fragment of the COI gene as described elsewhere [6]. The COI sequences of ticks from Xinjiang were 100% identical to A. persicus isolate E5 (OM368319.1), while the sequence of ticks from Heilongjiang was 100% identical to A. persicus isolate H1 (OM368320.1). Tick-borne agents, including Rickettsia, Anaplasmataceae bacteria, Coxiella, Borrelia, Bartonella, and Babesia, were screened using primers as previously shown [7, 8].

All the DNA samples were negative for Anaplasmataceae bacteria (including Ehrlichia, Anaplasma, and Neoehrlichia), Borrelia, Bartonella, and Babesia. Two Rickettsia species were detected and initially identified by amplifying and analyzing the partial 16S rDNA sequences (PCR products of approximately 900 bp) in A. persicus ticks from Heilongjiang, with positive rates of 27.3% (18/66) and 12.1% (8/66), respectively. Subsequently, the partial gltA (PCR products of approximately 1000 bp), groEL (PCR products of approximately 1100 bp), and ompB (PCR products of approximately 650 bp) genes were amplified and sequenced from randomly selected samples, as described elsewhere [7]. Based on the results, one Rickettsia species was determined as Rickettsia hoogstraalii, with the 16S rDNA, gltA, and ompB sequences showing 99.9%, 99.8%, and 99.4% identities to R. hoogstraalii str. RCCE3 or str. Croatica (LAOB01000001, LAOB01000002, EF629536). Interestingly, the other species was genetically distant from all known Rickettsia species. The 16S rDNA sequences had 98.8% identity to Rickettsia endosymbiont of the box bug Gonocerus acuteangulatus (OZ032147.1), 98.8% to Candidatus Rickettsia hubeiensis (OR979082.1), and 98.6% to Rickettsia bellii strain RML369-C (NR074484.2). Similarly, the groEL and ompB sequences had the highest identities 94.3% and 91.2%, to Candidatus Rickettsia hubeiensis (groEL: OR971720.1, OR971721.1; ompB: PP146537.1, PP146538.1). For the gltA gene, the rickettsia was closely related to the Rickettsia endosymbiont of Amblyomma patinoi with a nucleotide similarity of 93.1% (MN817133.1). In the phylogenetic trees constructed using PhyML 3.0 with the GTR + I + G model, all the strains formed a distinct clade together with R. bellii and Candidatus Rickettsia hubeiensis (Fig. 1), suggesting that it represents a novel species in the ancient group of the genus Rickettsia. The criteria of a novel Rickettsia species proposed by Fournier et al. [9] also support our opinion that it is a novel species. No Rickettsia was detected in ticks from Xinjiang.

Fig. 1

Phylogenetic trees based on the nucleotide sequences of 16S rDNA (809 bp), gltA (927–930 bp), groEL (1018 bp), and ompB (617 bp) genes of Rickettsia strains

Coxiella endosymbionts were detected using primers amplifying an approximately 550-bp conserved region of the rpoB gene [10]. All A. persicus ticks (positive rate 100%) were positive for Coxiella-like endosymbiont (CLE). However, in the phylogenetic tree based on rpoB sequences (Fig. 2), they were divided into two groups: one was only detected in ticks from Xinjiang (group 1), whereas the other was only detected in ticks from Heilongjiang (group 2). Both groups showed the highest nucleotide similarities to Coxiella sp. isolate EEZA-CRETAV2 identified in Argas sp. from Spain (MW287615.1) (96.1% and 98.7% nucleotide identities, respectively) and Coxiella burnetii (CP103427.1) (93. 9% and 95.6% nucleotide identities, respectively). For further exact identification of the CLEs, the DnaK sequence (PCR products of approximately 600 bp) was also obtained by nested PCR [10]. Similar to the rpoB gene, the DnaK sequences of CLE strains from ticks in Xinjiang and Heilongjiang were both closely related to Coxiella endosymbiont of Ornithodoros rostratus (KP985381.1) (with 96.2% and 97.1% nucleotide identities, respectively), Argas monachus (KP985358.1) (with 96.0% and 96.9% nucleotide identities, respectively), and Coxiella burnetii (ON455115.1) (with 95.2% and 95.4% nucleotide identities, respectively). In the phylogenetic tree, they also formed two distinct clades (Fig. 2).

Fig. 2

Phylogenetic trees based on the nucleotide sequences of rpoB (475 bp) and dnaK (560 bp) genes of Coxiella strains

In this study, two Rickettsia spp. were identified in A. persicus ticks from the same location in Heilongjiang Province: R. hoogstraalii and the novel species Rickettsia sp. Rickettsia hoogstraalii, belonging to the spotted fever group Rickettsia (SFGR), was first detected and isolated from Haemaphysalis sulcata in Croatia and Carios capensis in the USA, respectively [11]. Since then, it has been detected in multiple tick species (e.g., Argas transgariepinus, Argas walkerae, Rhipicephalus rossicus, Hyalomma anatolicum, Ornithodoros faini, Haemaphysalis formosensis) in several continents (Africa, Europe, Asia, and North America) [12,13,14,15]. In 2022, it was detected in A. persicus ticks from Gansu Province, Northwest China [16]. Our result confirmed the presence of R. hoogstraalii in China. Although it was only detected in ticks and never found in animals and humans, its close relationship with other SFGR members suggests its possible pathogenicity. Unexpectedly, a novel Rickettsia species belonging to the ancient group of genus Rickettsia was identified. In phylogenetic trees based on all genes investigated (i.e., 16S rDNA, gltA, groEL, and ompB), it was located in the basal position with R. bellii and Candidatus Rickettsia hubeiensis. Namely, it represents one of the several known members of the ancient group of Rickettsia. Until today, multiple Rickettsia species (e.g., Rickettsia japonica, R. raoultii, and R. rickettsii) have been identified in soft ticks [17, 18]. Of those, some were proposed as novel Rickettsia species. In 2018, a novel SFGR, Rickettsia fournieri, was identified in Argas lagenoplastis ticks in Australia [19]. As recently as 2025, a novel Rickettsia named "Candidatus Rickettsia vulgarisii" was described in Argas ticks from northwestern China [20]. However, almost all these Rickettsia spp. were SFGR, none belonging to the ancient group of Rickettsia. This result may contribute to knowledge of the genetic diversity of soft tick-borne Rickettsia.

CLE was detected in A. persicus ticks from both locations, with 100% positive rates, suggesting that CLE may be common in A. persicus ticks. Previous studies indicated that CLE is essential for B vitamin biosynthesis pathways in Rhipicephalus, Amblyomma, and Ornithodoros ticks [21]. Although the impact of CLE on the physiology of Argas ticks has never been studied, we suspect that it may play an important role in A. persicus considering the extremely high positive rates. Furthermore, it is interesting that the CLEs from different locations form distinct phylogenetic groups, indicating that one tick species can harbor different CLEs. They may have experienced long-term co-evolution together with their tick hosts. Besides, the A. persicus ticks from Xinjiang and Heilongjiang live in different habitats and have different animal hosts. They may have different sources of B vitamin intake. We suspect this might be one of the driving sources of CLE evolution.

In conclusion, we identified two Rickettsia species and two CLE species in A. persicus ticks from China. Of those, R. hoogstraalii belonging to SFGR may potentially infect animals and humans. Besides, although there are only a few reports that ancient group Rickettsia and CLEs infect animals or humans [22,23,24], the possibility remains. The presence of these agents in soft ticks may suggest a potential risk in these areas.