https://orcid.org/0009-0001-2046-3278
Figures
Abstract
Background
Tick-borne infectious diseases caused by the spotted fever group Rickettsia (SFGR) have continuously emerging, with many previously unidentified SFGR species reported. The prevalence of SFGRs in northwestern China remains unclear. This study aimed to examine the prevalence of SFGRs and Anaplasma species by analyzing tick samples collected from the Ningxia region.
Methods
During 2022–2023, ticks were collected from Ningxia, northwestern China, and screened using PCR to amplify target genes (16S rRNA, gltA, ompA and groEL). The amplicons were confirmed by Sanger sequencing. Single-gene sequences and concatenated sequences were used to infer phylogenetic relationships for identifying Rickettsia species.
Results
Out of the 425 DNA samples, a total of 210 samples tested positive for SFGRs in ticks from Ningxia, China, with a relatively high positive rate of 49.4% (210/425). Eight spotted fever group rickettsiae and one Anaplasma species were identified and characterized, including Rickettsia raoultii (102, 24.0%), R. aeschlimannii (65, 15.3%), R. sibirica (12, 2.8%), R. slovaca (4, 0.9%), R. heilongjiangensis (1, 0.2%), Cadidatus Rickettsia hongyuanensis (4, 0.9%), Ca. R. jingxinensis (11, 2.6%), Ca. R. vulgarisii (11, 2.6%) and Anaplasma ovis (98, 23.1%). The positive rate of bacterial species ranged from 0.2% to 24.0%. Interestingly, one novel Rickettsia species, provisionally named “Candidatus Rickettsia vulgarisii”, was detected in Argas ticks from Zhongwei city, which suggests the possibility of local transmission to other areas through birds. Genetic and phylogenetic analysis based on the 16S rRNA, gltA, ompA, and 17kDa genes indicated that it was divergent from all known SFG Rickettsia species but mostly related to R. vini. Different SFGR species were associated with specific tick species or genera. In addition, Anaplasma ovis was detected in two Dermacentor species, and co-infection with SFGRs was observed in 14.6% (62/425) of samples.
Conclusions
This study describes the prevalence and diversity of SFGRs in ticks from Ningxia for the first time by direct detection, reveals that Rickettsia diversity related to tick species. This data suggests that surveillance for tick-borne SFGR infections among human populations should be enhanced in this region, and further investigations on their pathogenicity to humans and domestic animals are still needed.
Author summary
SFGRs are bacteria mainly vectored by different tick species. There is increasing attention paid to them as causative agents of human disease. To date, these bacteria have been prevailing in China, but no studies have been conducted to detect potential tick vectors of rickettsiae in the Ningxia region. This study collected a total of 425 ticks from four cities with diverse geographic environments in Ningxia and determined the presence of SFGRs and Anaplasma spp. in naturally infected ticks. All the ticks were classfied as Argas, Dermacentor, Haemaphysalis s and Hyalomma genera. The overall prevalence of SFGRs and Anaplasma spp. detected in nine tick species were 49.4% and 23.1%, respectively. We present data that indicate high genetic diversity of SFGRs in Ningxia and co-infections with SFGR and A. ovis in ticks. Importantly, we describe a divergent Rickettsia spp. and propose the name Candidatus Rickettsia vulgarisii. These findings imply a potential threat of SFGRs infection among humans and highlight the importance of SFGRs and their targeted surveillance for human health in Ningxia.
Citation: Zhu W-J, Ye R-Z, Tian D, Wang N, Gao W-Y, Wang B-H, et al. (2025) The first direct detection of spotted fever group Rickettsia spp. diversity in ticks from Ningxia, northwestern China. PLoS Negl Trop Dis 19(1): e0012729. https://doi.org/10.1371/journal.pntd.0012729
Editor: Manisha Biswal, Postgraduate Institute of Medical Education and Research, INDIA
Received: July 31, 2024; Accepted: November 25, 2024; Published: January 2, 2025
Copyright: © 2025 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The sequences for the study are publicly available in the Genbank and can be accessed using accession numbers provided in S5 Table of this manuscript. Genbank accession number for SFGRs samples: PP110549–PP110758 (rrs), PP117689–PP117898 (ompA), and PP150126–PP150335 (gltA); For Anaplasma samples: PP106263–PP106360 (rrs), PP117399–PP117496 (groEL), and PP117094–PP117191 (gltA).
Funding: This work was supported by the National Key Research and Development Program of China (2021YFC2302001, X.-M. C.; 2023YFC2305901, D.-Y. Z), and the CAMS Innovation Fund for Medical Sciences (2019–I2M–5–049, W.-C. C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that they have no competing interests.
Introduction
Ticks are considered important arthropod vectors of various pathogens and have been associated with serious medical and veterinary health problems [1]. Many tick-borne bacterial agents are significant causes of unknown morbidity and mortality in human and domestic animals of great public health importance, e.g., spotted fever group rickettsiae (SFGRs), Anaplasma spp. and Coxiella spp. [2,3]. The majority of tick-borne infections are zoonotic, and their incidence and distribution are steadily increasing worldwide [4,5]. The distribution of various tick species and tick-borne agents in China has been studied for a long time. To date, at least 124 tick species from 9 genera have been reported across 1134 counties in China [6]. It was reported that more than 3,500 human cases had been confirmed infections with tick-borne pathogens (Borrelia spp., Anaplasma spp., Babesia spp. and SFGRs) covering the west, north, and northeast of China, including 32 cases of unexplained fever caused by Rickettsioses [6,7].
Rickettsia spp. and Anaplasma spp. belonging to the order Rickettsiales, of most concern in China, are common tick-borne bacterial pathogens. The SFGRs are obligate intracellular, gram-negative bacteria, generally associated with ticks, which can cause the emergence of spotted fever worldwide [8–11]. More than 20 species of SFGRs are causative agents of human diseases characterized by various clinical features, including fever, headache, rash, and cervical lymphadenopathy, which can be fatal in severe cases [12–15]. In mainland China, the emergence of SFGRs and corresponding rickettsial human cases have been predominantly reported in northeastern and central regions, where climatic conditions and human activities such as farming and livestock rearing favor the proliferation of tick populations, whereas in other areas, rickettsiosis is sporadic [16–20]. Several cases of rickettsial disease associated with acute fever and lymphadenopathy have been reported in Henan and Xinjiang regions of China [19,20]. In addition to humans, SFGRs infections have been detected in a variety of animal reservoir, including artiodactyla animals (sheep/goat/horse) [21,22], rodents [2], and wild birds [23]. Meanwhile, it is important to note that significant differences exist among tick species and their carried rickettsiae [24,25]. Anaplasma species have been also frequently detected in ticks and animals from multiple provinces in China [26]. Domestic animals, including sheep, cattle and goats, are often infected causing weight loss, reduced milk production, or even death, leading to great economic losses in animal husbandry annually [27]. Human infectious agents with these pathogens, such as A. phagocytophilum, A. capra and A. bovis, have been found in Inner Mongolia, Heilongjiang and Anhui provinces of China, but not as often as rickettsial cases [2,28–31].
Diverse manifestations of diseases can make their clinical diagnoses rather difficult. With the aid of molecular techniques, recent studies have expanded our knowledge on the diversity of vetor-tick bacteria and many novel species are being discovered globally with increasing frequency [31,32]. Some tick-borne bacteria species that were previously not considered pathogenic to humans are nowadays proven pathogens [33]. Diagnosis of infections with rickettsiae is commonly achieved by employing molecular biology-based analyses, specifically polymerase chain reaction (PCR) and nucleotide sequencing of DNA extracted from the patient [31,33]. Many different genes have been used for Rickettsia and Anaplasma phylogenetic systematics, including 16S rRNA, gltA, 17kDa, ompA, sca4, groEL, msp2 and complete genomic sequences by conventional, nested, and real-time PCR techniques [34]. However, relatively few investigations of tick-borne agents have been reported in the underdeveloped regions of northwestern China [35]. Thus, effective surveillance helps to determine tick populations, pathogen presence and seasonal activity, which is critical to implementing control measures. To be specific, investigations on the presence of tick-borne bacteria circulating in our environment within ticks—are of great medical and public health significance.
Located in northwestern China, Ningxia covers an area of 66,400 km2. The large topographic drop and the varied landform make for local complex ecological landscapes and diverse vegetation types. Combined with extensive livestock production activities, which is the major source of income for rural households, these conditions create a favorable environmental for tick growth and development [36,37]. Meanwhile, the frequent contact between villagers and domestic animals makes it possible for tick-borne pathogens and related diseases to be easily transmitted from animals or ticks to humans. The abundance of tick species varies substantially across diverse biogeographic zones defined by climatic and ecological characteristics. This study focuses on Ningxia, a region with distinct ecological and socio-economic characteristics that may promote tick development and tick-borne pathogen transmission. However, data about the prevalence and diversity on tick-borne bacteria in this area are limited, and relevant case studies are equally lacking. There have been no reports of SFGRs and Anaplasma species or even other pathogens directly detected from ticks. In this study, we collected ticks from five locations in Ningxia and analyzed the presence, prevalence, and genetic characteristics of the SFGRs and Anaplasma spp. in ticks using PCR and multi-locus sequence typing (MLST). Nucleotide sequence analysis and phylogenetic relationships are helpful to pathogen identification. Here we report the first finding of ticks, harbouring the pathogen SFGRs and Anaplasma spp. in Ningxia. The results of this study will be valuable in creating effective control measures to prevent zoonotic pathogens from spreading in this underexplored region.
Methods
Ethics statement
The collection of ticks from the body surface of host animals in this study was verbally consented by the animal owners and approved by the Animal Experiment Committee of the Laboratory Animal Center, Academy of Military Medical Sciences, China. The animal ethics approval number is IACUC-DWZX-027-20.
Sample collection and DNA extraction
Ticks were collected from livestock by using forceps, and vegetation surrounding the farms or living areas of animals by dragging white flags between March and May of the tick active period in 2022–2023 in all cities of Ningxia (38°27’58.9”N, 106°16’41.4”E), including Guyuan, Shizuishan, Wuzhong, Yinchuan, and Zhongwei city. Only adult ticks were identified and classified based on morphological criteria by an entomologist (Y.S.). Ticks were frozen and stored at −80°C until DNA extraction individually. We employed ArcGIS v10.8.2 to create detailed maps illustrating the geographical distribution of tick species across Ningxia region. The basemap shapefiles were downloaded from the Chinese Resource and Environmental Science Data Platform (http://www.resdc.cn/, DOI:10.12078/2023010102). These visualizations provide a clear spatial context for the prevalence of different tick species [38]. DNA extraction, amplification, and PCR product detection were carried out in separate rooms in order to prevent cross-contamination. Ticks were washed in distilled water for 10 min dried on sterile filter paper and homogenized individually with a single tick in Eppendorf tubes. Following the manufacturer’s instructions, the TaKaRa RNA/DNA Extraction Kit (TaKaRa, Dalian, China) was used for DNA extraction from homogenized ticks. Obtained total DNA was stored at –80°C.
PCR assays and sequencing
Ticks were examined for the presence of SFGRs and Anaplasma spp. by qualitative PCR (semi-nested and nested PCR) which amplify fragments of the 16S rRNA (rrs), outer membrane protein A gene (ompA), citrate synthase (gltA), and heat shock protein (groEL) genes [39,40]. Additionally, 17kDa gene was recovered for the putative novel Rickettsia species by nested PCR. PCR conditions comprised initial denaturation at 94°C for 3 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at temperatures, specified in S1 Table, for 30 sec and elongation at 72°C for 1.5 min. PCR primer sequences and conditions are listed in S1 Table. The DNA of R. raoultii and A. ovis were used for as positive control, whereas ddH2O was set as the negative control. PCR reactions were performed using a Veriti 96-Well Thermal Cycler (Applied Biosystems, Waltham, USA) and the PCR amplicons were subjected to Sanger sequencing in both directions after showing high intensity bands in 1.5% agarose gel electrophoresis. All the obtained nucleotide sequences were proofread, edited and assembled by CLC Main Workbench 5.0 (Qiagen, Redwood City, CA, USA)
Phylogenetic analysis
Samples that tested positive for all three genes of SFGRs or Anaplasma spp. were considered positive. Individual rrs, gltA, ompA or groEL sequence of the PCR products was compared to the sequences in the GenBank using the nucleotide Basic Local Alignment Search Tool (BLAST) [41]. Individual gene sequence and concatenated sequences were used for phylogenetic analysis. The assembled gene sequences were concatenated in the order of rrs, ompA (for Rickettsia)/groEL (for Anaplasma), and gltA. Additionally, reference sequences of different genes from various strains were obtained from GenBank, from which the amplified regions were extracted and concatenated in order (S2 Table). These sequences were aligned using MAFFT v7.505 [42] and adjusted using trimAl software [43]. All phylogenetic trees were constructed with maximum likelihood (ML) in IQ-TREE v2.2.0.3 with 1000 bootstrap replicates [44]. To further validate the evolutionary positions of gene sequences in newly discovered Rickettsia species, separate phylogenetic trees were constructed for the rrs, ompA, and gltA genes of Rickettsia species. These concatenated trees were annotated and visualized using the Tree Visualization by One Table online software [45].
Statistical analyses
The data obtained in this study were analyzed to estimate the proportion or percentage of SFGRs in different tick species with a 95% confidence interval (95% CI) including continuity correction based on one tick for a sample. Pearson’s chi-square (χ2) test or Fisher’s exact test was used to examine the differences in positive rates among tick species. Statistical significance was determined using GraphPad Prism 8 (GraphPad Software Inc., San Diego, California, USA); a P-value less than 0.05 was considered to indicate statistical significance. We used R, an open-source statistical programming platform, and the circlize package v0.4.16 to create chord diagrams to visualize the associations between tick species and the prevalence of Rickettsia species [46].
Results
Morphological identification of ticks
A total of 425 adult ticks were collected in four cities from Ningxia.After morphological identification, the ticks were classified into 4 genera and 9 species. These included Argas vulgaris (14, 3.29%), Dermacentor nuttalli (121 28.47%), Dermacentor silvarum (65, 15.29%), Haemaphysalis concinna (10, 2.35%), Haemaphysalis japonica (36, 8.47%), Haemaphysalis longicornis (42/425, 9.88%), Haemaphysalis qinghaiensis (36, 8.47%), Hyalomma asiaticum (24, 5.65%), and Hyalomma scupense (77, 18.12%). The distribution of tick species varies in different regions of Ningxia. (Fig 1). Ticks were collected in each city except Yinchuan (S3 Table). Dermacentor ticks (186/425, 43.8%) were the majority of ticks sampled and notably widespread, spanning the eastern, central, and southern regions of Ningxia (S1 Fig).
Different colour and size of circles represent the species and number of ticks collected from Ningxia, China. The map was constructed using ArcGIS v10.8.2 software. The basemap shapefiles were downloaded from the Chinese Resource and Environmental Science Data Platform (http://www.resdc.cn/, DOI:10.12078/2023010102).
Detection and characterization of Rickettsia spp.
We tested 425 adult ticks by nested or semi-nested PCR for the presence of SFGRs. In total, Rickettsial DNA were confirmed by Sanger sequencing in 210 (49.4%) of the 425 ticks. The gene sequences were subjected to BLAST analysis for a preliminary verification of their identity. BLAST results are shown in S4 Table. Phylogenetic trees based on the sequences of the rrs, gltA and ompA gene fragments with the ML method are shown in Fig 2. From the phylogenetic analysis based on three genes, most rickettsial sequences from this study clustered with R. raoultii and R. aeschlimannii isolates, some sequences of them were clustered together with R. sibirica, R. slovaca, R. heilongjiangensis, and Ca. R. hongyuanensis in branches, respectively. In addition, a few rickettsial sequences were most closely related to different SFGRs, even in separate clusters on these phylogenetic trees. For example, sample (TIGMIC125) clustered with R. sibirica (KU586293.1), R. africae (LC565700.1) and R. raoultii (MK304548.1) in the rrs, gltA and ompA phylogenetic trees, respectively (Fig 2).
The trees were constructed based on the nucleotide sequences of (a) rrs (760 bp), (b) gltA (381 bp) and (c) ompA (532 bp) using the maximum-likelihood method with the best substitution model found. All bootstrap support values from 1,000 replicates are shown at the interior branch nodes. The sequences obtained in this study are marked by orange circles.
Phylogeny of rickettsiae based on the concatenation of the rrs, ompA, and gltA genes
For further characterization of the detected bacterial strains, the sequences of ticks positive for all three genes (rrs, ompA and gltA) were concatenated and aligned for rickettsial phylogenetic analysis. The concatenated sequences also included validated Rickettsia species available in GenBank (S2 Table). In total, 210 tick DNA samples tested positive for Rickettsia were available for analysis. The validated Rickettsia sequences based on concatenated tree which overlooked these clade credibility values was delineated into the eight species of SFGRs, including six known species: R. raoultii, R. aeschlimannii, R. sibirica, R. slovaca, R. heilongjiangensis, and Ca. Rickettsia hongyuanensis (Fig 3).
The partial nucleotide sequences of genes rrs (760 bp), ompA (532 bp), and gltA (381 bp) were concatenated and constructed via the maximum-likelihood method by using the best substitution model found. A bootstrap analysis of 1,000 replicates was applied to assess the reliability of the reconstructed phylogenies. Study sample Rickettsia sp. Av11 and Rickettsia sp. DH11 branched distinctly from other rickettsiae and were considered novel SFGR genotypes. The various coloured lines represent distinct pathogens. Circles indicate host sources; solid circles denote samples from this study, while the rest represent reference sequences.
Among the ticks infected by Rickettsia species, the sequences of R. raoultii obtained in this study showed greater than 97.6% nucleotide (nt) identity with those of previously reported R. raoultii str. Khabarovsk isolated from D. silvarum in Russia and the R. raoultii isolate Datong-Dn-1 from D. nuttalli in China (Fig 3). R. aeschlimannii sequences had 99.7%–100% nt identity with multiple reference sequences (R. aeschlimannii isolates Baiyin-Ha-14, Baiyin-Hm-150, and RH15). The R. sibirica and R. slovaca strains detected in this study clustered together, comprising twelve and four positive samples, respectively. Sample sequences of R. sibirica shared more than 98.5% nt identity with R. sibirica str. RH05 isolated from Hya. truncatum in Senegal. Four sequences of R. slovaca identified in this study showed greater than 99.2% nt identity with R. slovaca strains 13-B and D-CWPP. R. heilongjiangensis was detected in only one sample (Hae. qinghaiensis), showing 99.3% nt identity with sequences from R. heilongjiangensis isolate XY-1 from Hae. longicornis in China.
Based on concatenated phylogenetic analysis, 22 samples from ticks with two novel SFGR genotypes with identical rrs, gltA and ompA gene sequences, which we designated Rickettsia sp. Av11 (TIGMIC196–206) and Rickettsia sp. DH11 (TIGMIC185–195). The sequences of 11 samples of Rickettsia sp. Av11 constituted an independent cluster on the concatenated tree, as a lone taxon between R. vini and Ca. R. jingxinensis, although showing exceeding 98.8% nt identity with the closest sequence from R. vini str. Boshoek1. Rickettsia sp. DH11. and Ca. R. jingxinensis comprise a separate cluster that appears most closely related to R. vini, R. japonica and R. heilongjiangensis (Fig 3). The rrs, ompA, and gltA sequences of SFGRs amplified from the tick samples were submitted to GenBank and assigned the accession numbers PP110549–PP110758 (rrs), PP117689–PP117898 (ompA), and PP150126–PP150335 (gltA) (S5 Table).
Comparative analysis of phylogenies in novel rickettsiae
In this study, we preliminary observed two possible new Rickettsia genotypes based on a phylogenetic tree constructed from concatenated sequences. To further describe the genetic characteristics of these new genotypes, single-gene segment phylogenetic trees were constructed based on the rrs, gltA, and ompA sequences of the new Rickettsia genotypes, and the results were comprehensively analyzed in conjunction with the concatenated sequence phylogenetic tree. Systematic analysis based on the rrs gene revealed a small genetic distance between branch sequences (S2 Fig), possibly due to the highly conservative nature of the rrs gene, which limits its effectiveness in species differentiation [47]. Therefore, we primarily referred to the phylogenetic trees based on the ompA and gltA genes and compared the two phylogenetic trees [48]. The sequences of Rickettsia sp. Av11 (TIGMIC196–206) in this study on the ompA phylogenetic tree were closest to R. vini (KX159440), showing 99.2%–99.4% identity, but formed an independent cluster on the gltA phylogenetic tree. A total of 11 sequences from Rickettsia sp. DH11 (TIGMIC185-195) were grouped together with Ca. R. jingxinensis (OQ702294) on the ompA phylogenetic tree, sharing 100% identity, whereas formed a separate branch on the gltA phylogenetic tree (Fig 4).
Phylogenetic analysis based on nucleotide sequences of two protein-encoding genes (on the left: ompA, on the left: gltA). The numbers at the nodes are bootstrap proportions with 1000 replicates. The scale bar indicates the number of nucleotide substitutions per site.
We amplified the 17kDa gene again with these samples from the possible new Rickettsia genotypes were compared with those available in GenBank. For the SFG Rickettsia sp. Av11, although the rrs gene was 100% identical to R. japonica str. LA16/2015 (CP047359.1), its 17kDa, ompA and gltA genes show 99.7%, 99.0%, 99.2% nucleotide similarity to R. heilongjiangensis isolate 2022-Tick251 (PP116500.1) from Hae. flava, R. vini str. Boshoek1 (MT062907.1) from Ixodes arboricola and Uncultured Rickettsia sp. isolate S6 (LC060714.1) from D. reticulatus, respectively. In the phylogenetic trees, its rrs and ompA genes were apparently divergent from other SFG Rickettsia species (Figs 3 and S2). Notably, its gltA gene is in a basal location in the phylogenetic tree but far from SFG Rickettsia species (Fig 3). According to the gene sequence-based criteria for taxonomic classification of new Rickettsia isolates, a Candidatus status could be assigned to Av11, so we named this species Candidatus Rickettsia vulgarisiii. For the Rickettsia sp. DH11, its rrs,ompA, gltA and 17kDa genes all show above 99.5% nucleotide similarity to Ca. R. jingxinensis (MH500194.1, MN463682.1, OR801782.1 and MW114879). Given that phylogenetic analysis of both rrs and ompA gene sequences revealed that the Rickettsia sp. DH11 strains were clustered with Ca. R. jingxinensis, so this SFG species was identified as Ca. R. jingxinensis.
Detection of Anaplasma spp.
To screen for Anaplasma infection in ticks, we also amplified the rrs, groEL, and gltA gene segments using nested PCR and detected 98 samples for all three positive genes. BLAST results are shown in S6 Table and phylogenetic trees based on single gene are shown in S3 Fig. As mentioned above, phylogenetic tree for Anaplasma species was also constructed by concatenated sequences from the three genes [49]. Among all positive sequences, 97 sample (TIGMIC001–097) showed 97.1%–100% identity to the Anaplasma ovis isolate TC249-5 detected in D. nuttalli from China, while the only remaining sample (TIGMIC098) fell between the two branches of A. ovis and A. capra (Fig 5), with 86.2% and 89.3% identity, respectively. Specific gene segments for A. ovis and A. capra were amplified again for this one sample, and only aligned with A. ovis (MH790273), showing 99.6% identity. This positive sample was identified as A. ovis. The nucleotide sequences of Anaplasma rrs, groEL, and gltA genes amplified from tick samples were submitted to GenBank with accession numbers PP106263–PP106360 (rrs), PP117399–PP117496 (groEL), and PP117094–PP117191 (gltA) (S5 Table).
The tree was based on concatenated rrs (660 bp), gltA (793 bp), and groEL (1100 bp) nucleotide sequences. The tree was constructed by the maximum-likelihood method and we performed bootstrap analysis of 1,000 replicates to assess the reliability of the constructed phylogenies. The numbers at the nodes are bootstrap proportions with 1000 replicates. The scale bar indicates the number of nucleotide substitutions per site. The sequences obtained in this study are marked by blue circles.
Prevalence of SFGRs and Anaplasma in Ticks
We screened 425 adult ticks from Ningxia region and Rickettsia and Anaplasma DNA were detected in 210 ticks and 98 ticks. The distribution of bacterial species and quantity is shown in S4 Fig. The most species and the highest positive number of SFGRs were in Guyuan city (65/210), while the highest positive rate was in Zhongwei city (68.8%, 53/77); the highest positive number and positive rate of Anaplasma species were in Guyuan city (50/98) and Wuzhong city (40.3%, 27/67), respectively (S4 Fig and S7 Table). We detected seven described species of Rickettsia and one novel candidate Rickettsia species (Figs 2–4). Sequencing of the Anaplasma amplicon determined the presence of one Anaplsma species in D. nuttalli, D. silvarum, Hae. longicornis and Hae. qinghaiensis (Fig 5). Eight species of Rickettsia were identified, while R. raoultii exhibited the highest infection rate (24.0%, 102/425), and R. heilongjiangensis had the lowest infection rate (0.2%, 1/425), detected solely in only one sample from Hae. qinghaiensis (Table 1). The prevalence of R. raoultii, the most abundant species, in Dermacentor ticks (52.7%, 98/186) was significantly higher than that in Haemaphysalis (3.2%, 4/124), Hyalomma (0/101), and Argas (0/14) ticks (χ2 = 149.6, df = 3, P < 0.001). R. aeschlimannii was exclusively detected in Hyalomma ticks (64.4%, 65/101), R. sibirica and R. slovaca were solely identified in Dermacentor ticks, Ca. R. hongyuanensis identified in Haemaphysalis ticks, whereas Ca. R. jingxinensis was detected in Dermacentor and Haemaphysalis ticks. The infection rates of the newly discovered SFGR was relatively high in Argas ticks (78.6%, 11/14). Furthermore, 98 samples tested positive for Anaplasma species, showing an overall positivity rate of 23.1% (95% CI: 19.1–27.1), all of which identified as A. ovis (Table 2). Anaplasma infection was detected only in Dermacentor and Haemaphysalis ticks (51.1%, 95/186, 2.4%, 3/124), with no evidence in other tick species. The infection rate of A. ovis in Dermacentor ticks was significantly higher than that in Haemaphysalis ticks and other tick genera (χ2 = 146.5, df = 3, P < 0.001). We identified the greatest richness of bacteria strains in D. nuttalli.
Co-infection of Rickettsia-Anaplasma within individual ticks was observed in 14.6% (62/425) of the ticks tested. Co-infections of R. raoultii + A. ovis were 11.5% (49/425), R. sibirica +A. ovis were 2.4% (10/425), and R. slovaca + A. ovis were 0.7% (3/425). All co-infected samples were Dermacentor ticks, with co-infection rate of 38.0% (46/121) in D. nuttalli and 24.6% (16/65) in D. silvarum (Table 3).
Vector host preference of rickettsiae
The above analysis results revealed differences in the parasitic adaptability of various Rickettsia species to tick hosts. To further describe this phenomenon, a species correlation analysis was conducted on all the samples and Rickettsia species included in this study. As illustrated in the chord diagram (Fig 6), these Rickettsia species exhibited specificity to tick genera or species. R. raoultii demonstrated relatively high adaptability to various tick species, being detected in three Haemaphysalis species and two Dermacentor species, with a positive rate reaching 60.3% in D. nuttalli, followed by D. silvarum (38.5%). R. aeschlimannii was found in both Hya. asiaticum and Hya. scupense, with positive rates of 50.0% and 68.8% (Table 1), respectively, and was not detected in tick genera other than Hyalomma ticks. R. sibirica and R. slovaca were exclusively found in Dermacentor ticks, Ca. R. vulgarisii was exclusively detected in Ar. vulgaris, whereas Ca. R. hongyuanensis was solely found in three species of Haemaphysalis ticks. While Ca. R. jingxinensis displayed a lower positive rate than R. raoulti, it was also detected in different tick species. These findings suggest that the distinct host preferences and adaptation ranges exhibited by different Rickettsia species.
The upper semicircle is the tick species, the lower semicircle is the Rickettsia species, and the color of the inner lines represent the tick species. The width of each chord corresponds to the count of Rickettsia-positive individuals in respective tick species.
Discussion
Rickettsia species exhibit broad pathogenicity, with some posing a lethal threat to humans [50], and ticks are critical vectors in the transmission cycle of these pathogens, affecting both human and animal health. Understanding the tick-host-pathogen interactions is essential for developing effective strategies to mitigate the impact of tick-borne diseases. Our results document the first detection in ticks of SFGRs and Anaplasma spp. collected from Ningxia, China. As a result, one Anaplasma species and eight SFGR species were identified in nine tick species. Sequence data provided evidence for the presence of A. ovis, R. raoultii, R. aeschlimannii, R. sibirica, R. slovaca, R. heilongjiangensis, Ca. R. hongyuanensis, Ca. R. jingxinensis, and one novel Rickettsia species, which we named “Ca. R. vulgarisii”.
SFGRs compose the important group in the genus Rickettsia. They are common tick-borne pathogens and have long been considered as the causative agents of various zoonotic diseases. In this study, a combination of single gene and concatenated genes (rrs, ompA, and gltA) were used to infer the evolutionary topology of Rickettsia using DNA samples obtained from ticks [51]. Eight Rickettsia species, especially a novel SFGR species named Ca. R. vulgarisii, were identified in ticks. Since its characterization in 2008 [52], R. raoultii, as a causative agent of human tick-borne lymphadenitis, has been reported in ticks from the Romania [24], India [53] and China [54]. The second SFGR identified in this study was R. aeschlimannii, which has been found in Hya. marginatum collected from southern Europe [55], and Africa [56]. R. sibirica, R. slovaca and R. heilongjiangensis are recognized as human pathogens that can cause mild rash associated with fever and eschars [57]. Ca. R. hongyuanensis and Ca. R. jingxinensis, belonging to SFG Rickettsia, were first identified in Hae. longicornis in southwest [58] and northeast China [59]. These findings indicate a human infection risk of important SFGR tick-borne diseases in the region.
Furthermore, we had detected an incompletely described Rickettsia in Argas ticks. This Rickettsia species had been detected with a highly positive rate in Zhongwei city, and had provisionally been named Rickettsia sp. Av11. The analysis of rrs, ompA and gltA genes and concatenated sequences confirmed the presence of this SFGR in Ningxia. As stated by de Sousa et al. [60], when they reported the detection of this bacterium, other gene sequences were required to establish its identity correctly according to the genetic guidelines published by Fournier et al. [61,62]. Genetic analyses indicate that its rrs, ompA, gltA and 17kDa genes have the highest identities to different validated species and these stains in different trees were apparently divergent from other SFG Rickettsia species. Considering these criteria, we propose to give to this strain a Candidatus status and name it “Ca. R. vulgarisii”, with reference to the name Ar. vulgaris ticks in which this Rickettsia species had been detected. Argas ticks can easily transmit and acquire bacteria to and from different hosts during its life cycle on account of the fact that they feed multiple times during a given developmental stage [63]. Wild birds, as the common hosts of Argas ticks, may be the non-negligible reason for introducing ticks and related pathogens into new environments due to their special migratory behaviors [64]. The role of Ar. vulgaris in the transmission of SFGRs should be further considered, and investigations on the human pathogenicity of these Rickettsia species are still needed. This discovery suggested that the potential threat of novel species to humans and animals still can not be excluded although its transmissibility and potential pathogenicity should be further studied.
A. ovis has been recognized as a tick-borne obligatory intraerythrocytic pathogen mainly infecting the ovine and caprine erythrocytes [65,66]. This pathogen can cause ovine anaplasmosis characterised by subclinical signs such as weakness, anorexia, weight loss and anaemia [67]. A. ovis was reported in all continents and has a widespread distribution in China [68], France [69] and Italy [70]. In this study, we observed remarkably high positivity rates of A. ovis in D. nuttalli (58.9%) and D. silvarum (45.7%). Hae. longicornis and Hae. qinghaiensis also tested positive for this A. ovis strain. Although the possibility cannot be ruled out that the A. ovis DNA may come from the blood meal of ticks, we suspect that Dermacentor ticks might play an important role in the transmission of A. ovis in domestic animals. The existence of A. ovis in Ningxia may indicate the risk of human infection and highlight the importance of surveillance in local populations though the pathogenicity of these strains to humans is still to be determined.
Evaluation of SFGRs and Anaplasma spp. prevalence at tick level was possible through phylogenetic analysis inference. The infection rates of SFGR and Anaplasma spp. in ticks from the Ningxia region were 49.4% and 23.1% in the present study and their positive rate among different tick species existed significance difference. The ticks were collected during the peak activity period of ticks, and Dermacentor ticks were the dominant tick genus, suggesting their important role in the distribution and transmission of rickettsiae in this region. The positive sequences of SFGRs and A. ovis in this study were mostly derived from ticks feeding on animal hosts (sheep and goat) rather than free ticks. These pathogens were found in regions with high biodiversity where migratory birds may inhabit. Combined with extensive livestock husbandry in Ningxia, they might spread to other hosts through these domestic animals. Previous studies have shown that mammals including sheeps and goats, particularly rodents and other small mammals, are often key reservoirs for tick-borne pathogens [2,21–23]. Meanwhile, studies have shown that a higher diversity of mammalian hosts can influence the transmission dynamics of these pathogens, reducing the transmission rates of pathogens like A. phagocytophilum [71]. This is due to the presence of non-susceptible species that can disrupt the cycle of transmission among more susceptible hosts. The ecological and economic factors in the Ningxia region have led to a rich array of animal resources. Therefore, according to the One health concept, it is not only human infections that are of concern, but infections in wild animals and livestock are also major health issues that deserve attention [72].
Our results also showed that co-infection of A. ovis and SFGR (R. raoultii, R. sibirica and R. slovaca) existed in Dermacentor ticks, and suggested potential interactions between these pathogens within tick vectors. The results obtained in the current study coincide with those previously described in Thailand, which reported co-infection with Rickettsia spp. and Anaplasma spp. in Dermacentor ticks [73]. It is reported that persistent infection of A. ovis can be accompanied by cyclical fluctuations in rickettsial levels, which may obviously alter the vector infection rate and thus alter transmission [74]. Previous research also indicated that co-infection often leads to a broader spectrum of clinical symptoms, prolongs disease duration, and exacerbates disease severity [75]. For instance, co-infection with both Borrelia burgdorferi and A. phagocytophilum, as opposed to single infections of either one, leads to more deep impairment of endothelial barrier function [76]. The higher co-infection rate in Dermacentor ticks, particularly D. nuttalli, suggests potential synergistic or competitive dynamics between these two genera of pathogens. The interactions between co-infections of pathogens within ticks and the impact of tick-borne transmission on human health still require further study [77].
Due to its intracellular lifestyle, rickettsiae are highly dependent on their primary tick vectors and tend to be selective for the tick species they infect. In this study, most bacteria were detected in Dermacentor ticks. Meanwhile, some bacteria were also detected in Haemaphysalis ticks, Hyalomma and Argas ticks. Further analysis of host preferences among different Rickettsia species highlighted differences in adaptability to various tick species. In this study, R. raoultii displayed relatively high adaptability across five tick species, with infections in Dermacentor ticks accounting for as much as 96.1% (98/102) of the tick population. In contrast, other Rickettsia species exhibited more specific host preferences. R. aeschlimannii showed a preference for two species of Hyalomma ticks. Among the newly discovered Rickettsia, Ca. R. vulgarisii was detected only in Ar. vulgaris, further emphasizing the genus-specific adaptation of Rickettsia species. Additionally, the detection of Anaplasma species in ticks also indicated that they have varying adaptability to different tick species. Beyond the Rickettsia and Anaplasma species studied herein, ticks generally engage in complex ecological interactions with their microbiota [78], where the presence of specific pathogens contributes to the stability of tick microbial communities and influences their population dynamics and metabolic functions [79,80]. The adaptability of Rickettsia species to specific hosts holds significant value in maintaining the structure and function of pathogen-vector microbial communities [81], suggesting that research on the mechanisms of co-evolution between ticks and the microbiota will enhance efforts to control the risk of Rickettsia disease transmission.
These findings presented here are of epidemiological importance. We have characterized a novel SFGR named “Ca. R. vulgarisii”, which may enhance our understanding of our knowledge on the diversity of SFGRs. Although the human pathogenicity for this novel bacterium from a limited sources of Argas ticks is still determined, more attention should be paid to the risk of human infection and the possible circulation of these pathogens in local population. Thus, further studies are needed to explore its implications for human and/or animal diseases in Ningxia by extending ecological surveys with an increased number of tick species and tick specimens. The host range, distribution, and pathogenicity of “Ca. R. vulgarisii” also merit further investigations.
Conclusions
In conclusion, the present study provided a molecular detection on Rickettsia spp. and Anaplasma spp. for the first time in ticks from Ningxia, northwestern China. One Anaplasma species (A. ovis) and egiht SFGR (R. raoultii, R. aeschlimannii, R. sibirica, R. slovaca, R. heilongjiangensis, Ca. R. hongyuanensis, Ca. R. jingxinensis and Ca. R. vulgarisii) including a novel species were detected and characterized. Our findings reveal a presence of diverse SFGRs including unidentified agents within tick species in Ningxia. Further investigations should be focused on expanding the sampling range, required to examine the effects of ecological and seasonal factors on ticks and pathogens, and ascertain the pathogenicity of newly emerged SFGRs in humans. Moreover, variations in host adaptability among different Rickettsia species highlight the complexity of the transmission and dissemination of tick-borne diseases. Therefore, there is a compelling need for intensified monitoring of tick and tick-borne pathogens in subsequent research attempt.
Supporting information
S1 Table. Nucleotide sequence of primers used for detecting ticks from Ningxia, China.
https://doi.org/10.1371/journal.pntd.0012729.s001
(DOCX)
S2 Table. GenBank accession numbers for validated strains used for concatenated sequence.
https://doi.org/10.1371/journal.pntd.0012729.s002
(DOCX)
S3 Table. Tick samples tested in this study and their location.
https://doi.org/10.1371/journal.pntd.0012729.s003
(DOCX)
S4 Table. Identity of positive sequence of detected Rickettsia spp. with BLAST analysis.
https://doi.org/10.1371/journal.pntd.0012729.s004
(DOCX)
S5 Table. Sequences of Rickettsia and Anaplasma from tick samples deposited in GenBank.
https://doi.org/10.1371/journal.pntd.0012729.s005
(DOCX)
S6 Table. Identity of positive sequence of Anaplasma spp. with BLAST analysis.
https://doi.org/10.1371/journal.pntd.0012729.s006
(DOCX)
S7 Table. Prevalence of rickettsiae and Anaplasma ovis in ticks from different cities of Ningxia.
https://doi.org/10.1371/journal.pntd.0012729.s007
(DOCX)
S1 Fig. Distribution of Dermacentor samples as the dominant tick species collected in Ningxia, China.
Different colour and size of circles represent the species and number of ticks collected from Ningxia. The map was constructed using ArcGIS v10.8.2 software. The basemap shapefiles were downloaded from the Chinese Resource and Environmental Science Data Platform (http://www.resdc.cn/, DOI:10.12078/2023010102).
https://doi.org/10.1371/journal.pntd.0012729.s008
(TIF)
S2 Fig. Phylogenetic analysis of unidentified Rickettsia (22 samples) based on the 760bp nucleotide sequence of rrs gene.
Numbers at the nodes are bootstrap proportions with 1000 replicates. The scale bar indicates the number of nucleotide substitutions per site.
https://doi.org/10.1371/journal.pntd.0012729.s009
(TIF)
S3 Fig. Phylogenetic trees of Anaplasma species based on the sequences of 3 different genes.
The trees were constructed based on the nucleotide sequences of (a) rrs (660 bp), (b) gltA (793 bp) and (c) groEL (1100 bp) using the maximum-likelihood method with the best substitution model found. All bootstrap support values from 1,000 replicates are shown at the interior branch nodes. The sequences obtained in this study are marked by blue circles.
https://doi.org/10.1371/journal.pntd.0012729.s010
(TIF)
S4 Fig. Distribution of Rickettsia and Anaplasma samples detected in Ningxia, China.
Different colour and size of circles (and cross symbols) represent the species and number of Rickettsia and Anaplasma detected from ticks. The map was constructed using ArcGIS v10.8.2 software. The basemap shapefiles were downloaded from the Chinese Resource and Environmental Science Data Platform (http://www.resdc.cn/, DOI:10.12078/2023010102).
https://doi.org/10.1371/journal.pntd.0012729.s011
(TIF)
Acknowledgments
Ningxia Medical University assisted with tick collection in Ningxia. We thank them for their help and constructive comments.
References
- 1. de la Fuente J, Estrada-Peña A, Rafael M, Almazán C, Bermúdez S, Abdelbaset AE, et al. Perception of ticks and tick-borne diseases worldwide. Pathogens. 2023;12(10):1258. pmid:37887774
- 2. Lu M, Li F, Liao Y, Shen JJ, Xu JM, Chen YZ, et al. Epidemiology and diversity of rickettsiales bacteria in humans and animals in Jiangsu and Jiangxi provinces, China. Sci Rep. 2019;9(1):13176. pmid:31511528
- 3. Ni J, Lin H, Xu X, Ren Q, Aizezi M, Luo J, et al. Coxiella burnetii is widespread in ticks (Ixodidae) in the Xinjiang areas of China. BMC Vet Res. 2020;16(1):317. pmid:32859190
- 4. Estrada-Peña A., De La Fuente J. The ecology of ticks and epidemiology of tick-borne viral diseases. Antivir. Res. 2014;108:104–128. pmid:24925264
- 5. Sanchez-Vicente S., Tagliafierro T., Coleman J.L., Benach J.L., Tokarz R. Polymicrobial nature of tick-borne diseases. Mbio. 2019;10:e02055–19. pmid:31506314
- 6. Zhao GP, Wang YX, Fan ZW, Ji Y, Liu MJ, Zhang WH, et al. Mapping ticks and tick-borne pathogens in China. Nat Commun. 2021;12(1):1075. pmid:33597544
- 7. Kang S, Zheng R. Distribution of the causes of fever of unknown origin in China, 2013–2022. J Transl Int Med. 2024;12(3):299–307. pmid:39081273
- 8. Osterloh A. The neglected challenge: vaccination against rickettsiae. PLoS Negl Trop Dis. 2020;14(10):e0008704. pmid:33091016
- 9. Diop A, El Karkouri K, Raoult D, Fournier PE. Genome sequence-based criteria for demarcation and definition of species in the genus Rickettsia. Int J Syst Evol Microbiol. 2020;70(3):1738–1750. pmid:31935173
- 10. Fournier PE, Raoult D. Current knowledge on phylogeny and taxonomy of Rickettsia spp. Ann NY Acad Sci. 2009;1166:1–11. pmid:19538259
- 11. Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005;18(4):719–756. pmid:16223955
- 12. Rieg S, Schmoldt S, Theilacker C, de With K, Wölfel S, Kern WV, et al. Tick-borne lymphadenopathy (TIBOLA) acquired in southwestern Germany. BMC Infect Dis. 2011;11:167. pmid:21663601
- 13. Cohen R, Finn T, Babushkin F, Paran Y, Ben Ami R, Atamna A, et al. Spotted fever group rickettsioses in Israel, 2010–2019. Emerg Infect Dis. 2021;27(8):2117–2126. pmid:34286684
- 14. Walker DH, Valbuena GA, Olano JP. Pathogenic mechanisms of diseases caused by Rickettsia. Ann N Y Acad Sci. 2003;990:1–11. pmid:12860594
- 15. Boulanger N, Boyer P, Talagrand-Reboul E, Hansmann Y. Ticks and tick-borne diseases. Med Mal Infect. 2019;49(2):87–97. pmid:30736991
- 16. Chen Z, Yang X, Bu F, Yang X, Yang X, Liu J. Ticks (acari: ixodoidea: argasidae, ixodidae) of China. Exp Appl Acarol. 2010;51(4):393–404. pmid:20101443
- 17. Yu Z, Wang H, Wang T, Sun W, Yang X, Liu J. Tick-borne pathogens and the vector potential of ticks in China. Parasit Vectors. 2015;8:24. pmid:25586007
- 18. Zhang JZ, Fan MY, Wu YM, Fournier PE, Roux V, Raoult D. Genetic classification of "Rickettsia heilongjiangii" and "Rickettsia hulinii," two Chinese spotted fever group rickettsiae. J Clin Microbiol. 2000;38(9):3498–3501. pmid:10970415
- 19. Liu W, Li H, Lu QB, Cui N, Yang ZD, Hu JG, et al. Candidatus Rickettsia tarasevichiae infection in eastern central China: a case series. Ann Intern Med. 2016;164(10):641–648. pmid:27019406
- 20. Fan MY, Wang JG, Jiang YX, Zong DG, Lenz B, Walker DH. Isolation of a spotted fever group Rickettsia from a patient and related ecologic investigations in Xinjiang Uygur Autonomous Region of China. J Clin Microbiol. 1987;25(4):628–632. pmid:3571471
- 21. Ma Y, Jian Y, Wang G, Zafar I, Li X, Wang G, et al. Epidemiological investigation of tick-borne bacterial pathogens in domestic animals from the Qinghai-Tibetan Plateau Area, China. Pathogens. 2024;13(1):86. pmid:38276159
- 22. Chang QC, Hu Y, Wu TT, Ma XX, Jiang BG, Jia N, et al. The role of ranged horses in eco-epidemiology of Rickettsia raoultii infection in China. Front Microbiol. 2022;12:795500. pmid:35111141
- 23. Hoffman T, Sjödin A, Öhrman C, Karlsson L, McDonough RF, Sahl JW, et al. Co-occurrence of Francisella, spotted fever group Rickettsia, and Midichloria in Avian-associated Hyalomma rufipes. Microorganisms. 2022;10(7):1393. pmid:35889112
- 24. Ivan T, Matei IA, Novac CȘ, Kalmár Z, Borșan SD, Panait LC, et al. Spotted fever group Rickettsia spp. diversity in ticks and the first report of Rickettsia hoogstraalii in Romania. Vet Sci. 2022;9(7):343. pmid:35878360
- 25. Merhej V, Angelakis E, Socolovschi C, Raoult D. Genotyping, evolution and epidemiological findings of Rickettsia species. Infect Genet Evol. 2014;25:122–37. pmid:24662440
- 26. Wang H, Yang J, Mukhtar MU, Liu Z, Zhang M, Wang X. Molecular detection and identification of tick-borne bacteria and protozoans in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. Parasit Vectors. 2019;12(1):296. pmid:31196180
- 27. Parvizi O, El-Adawy H, Roesler U, Neubauer H, Mertens-Scholz K. Performance analysis of Anaplasma antibody competitive ELISA using the ROC curve for screening of Anaplasmosis in camel populations in Egypt. Pathogens. 2020;9(3):165. pmid:32121019
- 28. Tsai KH, Chung LH, Chien CH, Tung YJ, Wei HY, Yen TY, et al. Human granulocytic anaplasmosis in Kinmen, an offshore island of Taiwan. PLoS Negl Trop Dis. 2019;13(9):e0007728. pmid:31539395
- 29. Gaowa Wulantuya, Yin X, Cao M, Guo S, Ding C, et al. Case of human infection with Anaplasma phagocytophilum in Inner Mongolia, China. Jpn J Infect Dis. 201871(2):155–157. pmid:29491236
- 30. Li H, Zheng YC, Ma L, Jia N, Jiang BG, Jiang RR, et al. Human infection with a novel tick-borne Anaplasma species in China: a surveillance study. Lancet Infect Dis. 2015;15(6):663–670. pmid:25833289
- 31. Lu M, Chen Q, Qin X, Lyu Y, Teng Z, Li K, et al. Anaplasma bovis infection in fever and thrombocytopenia patients—Anhui Province, China, 2021. China CDC Wkly. 2022;4(12):249–253. pmid:35433083
- 32. Peckle M, Luz HR, Labruna MB, Serpa MCA, Lima S, Maturano R, et al. Multi-locus phylogenetic analysis groups the New World bacterium Rickettsia sp. strain ApPR with the Old World species R. africae; proposal of "Candidatus Rickettsia paranaensis". Ticks Tick Borne Dis. 2019;10(6):101261. pmid:31337544
- 33. Jia N, Jiang JF, Huo QB, Jiang BG, Cao WC. Rickettsia sibirica subspecies sibirica BJ-90 as a cause of human disease. N Engl J Med. 2013;369(12):1176–1178. pmid:24047079
- 34. Stewart AG, Stewart AGA. An update on the laboratory diagnosis of Rickettsia spp. Infection. Pathogens. 2021;10(10):1319. pmid:34684267
- 35. Zhang G, Zheng D, Tian Y, Li S. A dataset of distribution and diversity of ticks in China. Sci Data. 2019;6(1):105. pmid:31263100
- 36. Tagliapietra V, Rosà R, Arnoldi D, Cagnacci F, Capelli G, Montarsi F, et al. Saturation deficit and deer density affect questing activity and local abundance of Ixodes ricinus (Acari, Ixodidae) in Italy. Vet Parasitol. 2011;183(1):114–124. pmid:21820245
- 37. Titcomb G, Allan BF, Ainsworth T, Henson L, Hedlund T, Pringle RM, et al. Interacting effects of wildlife loss and climate on ticks and tick-borne disease. Proc Biol Sci. 2017;284(1862):20170475. pmid:28878055
- 38. Zhao L, Li J, Cui X, Jia N, Wei J, Xia L, et al. Distribution of Haemaphysalis longicornis and associated pathogens: analysis of pooled data from a China field survey and global published data. Lancet Planet Health. 2020;4(8):e320–e329. pmid:32800150
- 39. Li H, Li XM, Du J, Zhang XA, Cui N, Yang ZD, et al. Candidatus Rickettsia xinyangensis as cause of spotted fever group rickettsiosis, Xinyang, China, 2015. Emerg Infect Dis. 2020;26(5):985–988. pmid:32310072
- 40. Lin ZT, Du LF, Zhang MZ, Han XY, Wang BH, Meng J, et al. Genomic characteristics of emerging intraerythrocytic Anaplasma capra and high prevalence in goats, China. Emerg Infect Dis. 2023;29(9):1780–1788. pmid:37610104
- 41. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–410. pmid:2231712
- 42. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–780. pmid:23329690
- 43. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. TrimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–1973. pmid:19505945
- 44. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268–274. pmid:25371430
- 45. Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res. 2023;51(W1):W587–W592. pmid:37144476
- 46. Gu Z, Gu L, Eils R, Schlesner M, Brors B. Circlize Implements and enhances circular visualization in R. Bioinformatics. 2014;30(19):2811–2812. pmid:24930139
- 47. Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA. 2009;106:19126–19131. pmid:19855009
- 48. Thomas GH. Evolution: an avian explosion. Nature. 2015;526(7574):516–517. pmid:26444233
- 49. Karpathy SE, Kingry L, Pritt BS, Berry JC, Chilton NB, et al. Anaplasma bovis-like infections in humans, United States, 2015–2017. Emerg Infect Dis. 2023;29(9):1904–1907. pmid:37610264
- 50. Guccione C, Colomba C, Iaria C, Cascio A. Rickettsiales in the WHO European Region: an update from a One Health perspective. Parasit Vectors. 2023;16(1):41. pmid:36717936
- 51. Kimita G, Mutai B, Nyanjom SG, Wamunyokoli F, Waitumbi J. Phylogenetic variants of Rickettsia africae, and incidental identification of "Candidatus Rickettsia Moyalensis" in Kenya. PLoS Negl Trop Dis. 2016;10(7):e0004788. pmid:27387337
- 52. Mediannikov O, Matsumoto K, Samoylenko I, Drancourt M, Roux V, Rydkina E, et al. Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia. Int J Syst Evol Microbiol. 2008;58(Pt 7):1635–1639.
- 53. Nallan K, Ayyavu V, Ayyanar E, Thirupathi B, Gupta B, Devaraju P, et al. Molecular evidence of Rickettsia conorii subsp. raoultii and Rickettsia felis in Haemaphysalis intermedia ticks in Sirumalai, Eastern Ghats, Tamil Nadu, South India. Microorganisms. 2023;11(7):1713. pmid:37512886
- 54. Cheng C, Fu W, Ju W, Yang L, Xu N, Wang YM, et al. Diversity of spotted fever group Rickettsia infection in hard ticks from Suifenhe, Chinese-Russian border. Ticks Tick Borne Dis. 2016;7(5):715–719. pmid:26976703
- 55. Matsumoto K, Parola P, Brouqui P, Raoult D. Rickettsia aeschlimannii in Hyalomma ticks from Corsica. Eur J Clin Microbiol Infect Dis. 2004;23(9):732–734. pmid:15309667
- 56. Pretorius AM, Birtles RJ. Rickettsia aeschlimannii: A new pathogenic spotted fever group rickettsia, South Africa. Emerg Infect Dis. 2002;8(8):874. pmid:12141981
- 57. Sayfullin RF, Perekopskaya NE, Karan LS, Zvereva NN, Sayfullin MA. Autochthonous case of Rickettsia slovaca infection in Russia. Emerg Infect Dis. 2021;27(10):2736–2738. pmid:34546169
- 58. Lu M, Tian J, Zhao H, Jiang H, Qin X, Wang W, et al. Molecular survey of vector-borne pathogens in ticks, sheep keds, and domestic animals from Ngawa, southwest China. Pathogens. 2022;11(5):606. pmid:35631127
- 59. Liu H, Li Q, Zhang X, Li Z, Wang Z, Song M, et al. Characterization of rickettsiae in ticks in northeastern China. Parasit Vectors. 2016;9(1):498. pmid:27623998
- 60. De Sousa R, Barata C, Vitorino L, Santos-Silva M, Carrapato C, Torgal J, et al. Rickettsia sibirica isolation from a patient and detection in ticks, Portugal. Emerg Infect Dis. 2006;12:1103–1108. pmid:16836827
- 61. Raoult D, Fournier PE, Eremeeva M, Graves S, Kelly PJ, Oteo JA, et al. Naming of rickettsiae and rickettsial diseases. Ann NY Acad Sci. 2005;1063:1–12. pmid:16481485
- 62. Fournier PE, Dumler JS, Greub G, Zhang JZ, Wu YM, Raoult D. Gene sequence-based criteria for identification of new Rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov. J Clin Microbiol. 2003;41:5456–5465. pmid:14662925
- 63. Narasimhan S, Fikrig E. Tick microbiome: the force within. Trends Parasitol. 2015;31(7):315–323. pmid:25936226
- 64. Bertelloni F, Cagnoli G, Interrante P, Ceccherelli R, Ebani VV. Molecular survey on the occurrence of tick-borne bacteria in wild birds from central Italy. Vet Sci. 2024;11(7):284. pmid:39057968
- 65. Ruiz H, de Arcaute MR, Benito AÁ, Villanueva-Saz S, Jiménez JC, Lacasta D. Long-lasting infection with Anaplasma ovis in sheep. Vet Res Commun. 2024;48(1):521–525. pmid:37528260
- 66. Pereira A, Parreira R, Nunes M, Casadinho A, Vieira ML, Campino L, et al. Molecular detection of tick-borne bacteria and protozoa in cervids and wild boars from Portugal. Parasit Vectors. 2016;9(1):251. pmid:27160767
- 67. Lacasta D, Lorenzo M, González JM, Ruiz de Arcaute M, Benito AÁ, Baselga C, et al. Epidemiological study related to the first outbreak of ovine anaplasmosis in Spain. Animals (Basel). 2021;11(7):2036. pmid:34359164
- 68. Zhao L, He B, Li KR, Li F, Zhang LY, Li XQ, et al. First report of Anaplasma ovis in pupal and adult Melophagus ovinus (sheep ked) collected in South Xinjiang, China. Parasit Vectors. 2018;11(1):258. pmid:29673387
- 69. Cabezas-Cruz A, Gallois M, Fontugne M, Allain E, Denoual M, Moutailler S, et al. Epidemiology and genetic diversity of Anaplasma ovis in goats in Corsica, France. Parasit Vectors. 2019;12(1):3. pmid:30606253
- 70. André MR. Diversity of Anaplasma and Ehrlichia/Neoehrlichia agents in terrestrial wild carnivores worldwide: implications for human and domestic animal health and wildlife conservation. Front Vet Sci. 2018;5:293. pmid:30533417
- 71. Fabri ND, Heesterbeek H, Cromsigt JPGM, Ecke F, Sprong H, Nijhuis L, et al. Exploring the influence of host community composition on the outbreak potential of Anaplasma phagocytophilum and Borrelia burgdorferi s.l. Ticks Tick Borne Dis. 2024;15(1):102275. pmid:37922668
- 72. Machtinger ET, Poh KC, Pesapane R, Tufts DM. An integrative framework for tick management: the need to connect wildlife science, One Health, and interdisciplinary perspectives. Curr Opin Insect Sci. 2024;61:101131. pmid:37866434
- 73. Nooroong P, Trinachartvanit W, Baimai V, Ahantarig A. Phylogenetic studies of bacteria (Rickettsia, Coxiella, and Anaplasma) in Amblyomma and Dermacentor ticks in Thailand and their co-infection. Ticks Tick Borne Dis. 2018;9(4):963–971. pmid:29610046
- 74. Palmer GH, Abbott JR, French DM, McElwain TF. Persistence of Anaplasma ovis infection and conservation of the msp-2 and msp-3 multigene families within the genus Anaplasma. Infect Immun. 1998;66(12):6035–6039. pmid:9826393
- 75. Cutler SJ, Vayssier-Taussat M, Estrada-Peña A, Potkonjak A, Mihalca AD, Zeller H. Tick-borne diseases and co-infection: current considerations. Ticks Tick Borne Dis. 2021;12(1):101607. pmid:33220628
- 76. Grab DJ, Nyarko E, Barat NC, Nikolskaia OV, Dumler JS. Anaplasma phagocytophilum-Borrelia burgdorferi coinfection enhances chemokine, cytokine, and matrix metalloprotease expression by human brain microvascular endothelial cells. Clin Vaccine Immunol. 2007;14(11):1420–1424. pmid:17898182
- 77. Hoffmann A, Müller T, Fingerle V, Silaghi C, Noll M. Co-infection of potential tick-borne pathogens of the order Rickettsiales and Borrelia burgdorferi s. l. and their link to season and area in Germany. Microorganisms. 2023;11(1):157. pmid:36677449
- 78. Wu-Chuang A, Obregon D, Estrada-Peña A, Cabezas-Cruz A. Thermostable keystone bacteria maintain the functional diversity of the Ixodes scapularis microbiome under heat stress. Microb Ecol. 2022;84(4):1224–1235. pmid:34817640
- 79. Maitre A, Wu-Chuang A, Mateos-Hernández L, Piloto-Sardiñas E, Foucault-Simonin A, Cicculli V, et al. Rickettsial pathogens drive microbiota assembly in Hyalomma marginatum and Rhipicephalus bursa ticks. Mol Ecol. 2023;32(16):4660–4676. pmid:37366236
- 80. Berg G, Rybakova D, Fischer D, Cernava T, Vergès MC, Charles T, et al. Microbiome definition re-visited: old concepts and new challenges. Microbiome. 2020;8(1):103. pmid:32605663
- 81. Maitre A, Wu-Chuang A, Aželytė J, Palinauskas V, Mateos-Hernández L, Obregon D, et al. Vector microbiota manipulation by host antibodies: the forgotten strategy to develop transmission-blocking vaccines. Parasit Vectors. 2022;15(1):4. pmid:34983601