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Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39193640
2396870
10.1080/22221751.2024.2396870
Version of Record
Research Article
Research Article
Genomic characterization of an emerging Rickettsia barbariae isolated from tick eggs in northwestern China
EMERGING MICROBES & INFECTIONS
N. WANG ET AL.
Wang Ning ab†
Yu Hui-Jun a†
Han Xiao-Yu b
Li Cheng a
Ye Run-Ze c
Du Li-Feng a
Liu Ya-Ting b
Zhang Ming-Zhu a
Shi Xiao-Yu b
Zhu Dai-Yun b
Shi Wenqiang b
Jia Na b
Jiang Jia-Fu b
Sun Yi b
Zhao Lin a*
Cui Xiao-Ming b*
Cao Wu-Chun ab*
a Institute of EcoHealth, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, People’s Republic of China
b State Key Laboratory of Pathogen and Biosecurity, AMMS, Beijing, People’s Republic of China
c Department of Emergency Medicine, Qilu Hospital of Shandong University, Jinan, People’s Republic of China
CONTACT Wu-Chun Cao caowuchun@126.com
Xiao-Ming Cui cuixm7@163.com
Lin Zhao zhaolin1989@sdu.edu.cn
* These authors contributed equally to this article.

† These senior authors contributed equally to this article.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2396870.

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The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

The continual emergence of tick-borne rickettsioses has garnered widespread global attention. Candidatus Rickettsia barbariae (Candidatus R. barbariae), which emerged in Italy in 2008, has been detected in humans from northwestern China. However, the lack of Candidatus R. barbariae genome and isolated strains limits the understanding of its biological characteristics and genomic features. Here, we isolated the Rickettsia for the first time from eggs of Rhipicephalus turanicus in northwestern China, and assembled its whole genome after next-generation sequencing, so we modified the proposed name to Rickettsia barbariae (R. barbariae) to conform to the International Code of Nomenclature of Prokaryotes. Phylogenetic analysis based on the whole genome revealed that it was most closely related to the pathogenic Rickettsia parkeri and Rickettsia africae. All virulence factors, present in the pathogenic spotted fever group rickettsiae, were identified in the R. barbariae isolate. These findings highlight the pathogenic potential of R. barbariae and the necessity for enhanced surveillance of the emerging Rickettsia in the human population.

KEYWORDS

Rickettsia barbariae
Rhipicephalus turanicus
whole-genome sequencing
phylogenetic analysis
virulence factors
China
National Key Research and Development Program of China 10.13039/501100012166 2023YFC2305901 Natural Science Foundation of China 10.13039/501100001809 81621005 Postdoctoral Fellowship Program of CPSF BX20240215 This work was supported by National Key Research and Development Program of China: [Grant Number 2023YFC2305901]; Natural Science Foundation of China: [Grant Number 81621005]; Postdoctoral Fellowship Program of CPSF: [Grant Number BX20240215].
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pmcIntroduction

The continual emergence of tick-borne rickettsioses has garnered widespread global attention [1–3]. The isolation and characterization of emerging Rickettsia are of significant importance for the surveillance and control of tick-borne diseases. Candidatus Rickettsia barbariae (Candidatus R. barbariae) was initially identified by polymerase chain reaction (PCR) in Rhipicephalus turanicus (Rh. turanicus) from Italy in 2008 [4]. The emerging Rickettsia has been detected not only in Mediterranean countries but also in the United States, Central Asia, and Xinjiang of China. Rhipicephalus and Hyalomma ticks [5–10] are common vectors for this Rickettsia, and some wild animals [11,12] have been reported to be infected with it. Additionally, related gene fragments have been detected in humans by PCR, suggesting potential pathogenicity to humans. However, the absence of isolates and whole-genome sequences limits the understanding of its biological characteristics and genomic features.

Considering the characteristic transovarial transmission of most spotted fever group rickettsiae (SFGR) [13,14], we conducted molecular detection of SFGR on the eggs laid by engorged Rh. turanicus ticks and isolated those testing positive in cell culture. Following successful isolation, we sequenced and assembled the complete genome of the emerging Rickettsia, identified its genomic characteristics, and designated it as Rickettsia barbariae strain BIME (R. barbariae str. BIME).

Materials and methods

Tick collection, DNA extraction, and PCR screening

Adult Rh. turanicus ticks were collected from goats in Xinjiang, China (81°2′31″E, 44°28′42″N). Each engorged female tick was individually placed in a 15 mL centrifuge tube to lay eggs, and the ticks were reared under a 12-hour light/12-hour dark photoperiod at a temperature of 25°C in an artificial climate incubator with 95% relative humidity. DNA was extracted from each pool of eggs (∼100 eggs/pool) or ticks using the MiniBEST Viral RNA/DNA Extraction Kit (Takara, Japan) according to the manufacturer’s instructions. Rickettsia was subsequently tested using specific PCR assays targeting the ompA, gltA, ompB, sca4, and sca1 genes [15–18], followed by Sanger sequencing to identify the Rickettsia species (Table S1).

Isolation, purification, and DNA extraction of Rickettsia

A pool of Rickettsia-positive Rh. turanicus eggs was used for isolation. The pooled eggs were ground and the supernatant was inoculated into Ixodes scapularis (I. scapularis) tick cell line IDE8 in 24-well culture plates. Giemsa staining and quantitative real-time polymerase chain reaction (qPCR) targeting the sca1 gene (Table S1) [19] were used for assessing the isolation of Rickettsia every two weeks to observe the growth dynamics. The copy number of Rickettsia DNA at each time point was calculated using a standard curve to obtain the growth curve. The standard curve formula was Y = −0.2751X + 11.362, where X was the cycle threshold (Ct) value measured by quantitative fluorescence, Y represented the copy number in log10, and the copy number was expressed in copies/µL. The culture was harvested from the wells when the Ct value was below 15, and transferred to 25 cm2 flasks for further proliferation. Once the qPCR Ct value came to 15, we collected the cell culture and blew the cells repeatedly using a 27G needle to release the Rickettsia. We then centrifuged the mixture at 1,000 ×g for 10 minutes to get rid of cell debris. The supernatant was centrifuged at 17,000 ×g for 10 minutes at 4°C to enrich the Rickettsia. Total DNA was extracted from the purified Rickettsia using the MiniBEST Universal Genomic DNA Extraction Kit (Takara, Japan) for next-generation sequencing.

Whole-genome sequencing and assembly

The total DNA was subjected to next-generation sequencing using the RK20208-Rapid Plus DNA Lib Prep Kit for Illumina (ABclonal, China) and the Illumina NovaSeq 6000 platform with a read length of 2 × 150 base pairs (bp) at Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Cleaned sequence reads were mapped to the genome of I. scapularis (GenBank accession no. GCF_016920785.2) by Bowtie2 v2.4.1 [20] with default options for eliminating the host genome. The remaining reads, which were not matched to the I. scapularis genome, were converted to BAM files by SAMtools v1.9 [21]. Host-free reads were assembled into contigs and scaffolds using SPAdes v3.15.5 [22]. MetaBAT2 v2:2.15 [23] was used for contig binning. Prokka v1.13.3 [24] and CheckM v1.1.3 [25] with the lineage_wf argument were used for genome annotation and completeness assessment. Average nucleotide identity (ANI) was estimated using fastANI v1.32 [26].

Phylogenetic analyses

A multiple sequence comparison based on the whole genome of R. barbariae and the genomes of SFGR deposited in GenBank was performed using the software PhyloPhlAn v3.0.67 [27]. The maximum likelihood phylogenetic tree was constructed using IQ-Tree v2.2.0.3 [28]. Furthermore, the Rickettsia ompA, ompB, gltA, sca1, and sca4 genes were extracted from the R. barbariae genome using Blast + v2.13.0 [29] after matching with reference genes and combining with Prokka annotation results. These genes were then aligned with reference sequences obtained from GenBank using MAFFT v7.505 [30], and poorly aligned parts were trimmed using TrimAl v1.4.rev15 [31]. Phylogenetic analyses based on the five conserved genes were conducted respectively by using the maximum likelihood method in IQ-Tree v2.2.0.3. All phylogenetic trees were visualized using the ggtree package in R software [32].

Functional analysis of predicted genes

Orthofinder v2.5.4 [33] was utilized to find single-copy orthologue sequences and orthogroups, and UpsetR [34] was used to visualize shared, unique, and intersection situations in orthogroups. EggNOG-mapper v2.1.7 [35] was used for function annotation of the whole genome of R. barbariae and selected reference genomes from GenBank. The functions of predicted protein-coding sequences (CDSs) were identified by searching against the Clusters of Orthologous Groups of proteins (COG), eggNOG, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. This analysis used an E-value threshold of 1e−5 and was followed by filtering to retain only the best match.

Results

Identification, genomic, and taxonomic characterization of R. barbariae

In May 2023, six engorged female Rh. turanicus ticks were collected from Wujiaqu City, Xinjiang, China, and reared to facilitate egg laying (Figure S1). We screened their eggs for SFGR by specific PCRs targeting the ompA and gltA genes, followed by Sanger sequencing. Among them, one pool of eggs tested positive for SFGR, with its gltA gene and ompA gene sequences (GenBank accession nos. PP954849 and PP954852) showing 100.00% homology (368/368 and 507/507) with Candidatus R. barbariae isolates SHZ-50 and SHZ (GenBank accession nos. MW802692 and KY069256) from Rh. turanicus in Xinjiang, China. Based on these findings, we preliminarily identified this strain of Rickettsia as Candidatus R. barbariae and attempted to isolate and culture it.

We inoculated the positive eggs of Rh. turanicus onto IDE8 cells and replaced the medium weekly. Using qPCR targeting the sca1 gene, we found that the bacterial load decreased from 105 upon inoculation to 103.6, but gradually increased to 107 after five weeks of cell adaptation (Figure 1A). The decline in the bacterial copies in the initial stage might be attributed to the time required for Rickettsia to adapt to the cell before proliferation. By week 13, Giemsa staining revealed the presence of rickettsial bacilli, appearing as deeply stained, short-rod, long-rod, or spherical bodies in shades of blue-purple, proliferating significantly and causing cellular pathology, eventually releasing from the cytoplasm (Figure 1B). In IDE8 cells examined by transmission electron microscopy, Rickettsia were scattered in the cytoplasm and appeared as coccoid or bacillary bacteria in the cytosol (Figure 1C). Figure 1. Isolation and fundamental genomic information of R. barbariae str. BIME. (A) Growth curves of R. barbariae in IDE8 tick cells over 13 weeks after inoculated the eggs of Rh. turanicus onto IDE8 cells. (B) Giemsa staining of R. barbariae isolated from the eggs of Rh. turanicus in IDE8 cells. Rickettsia appeared as deeply stained, short-rod, long-rod, or spherical bodies in shades of blue-purple. The uninfected IDE8 cells cultivated in parallel were used as controls. Scale bar represents 10 μm. (C) Transmission electron micrographs of IDE8 cells infected with R. barbariae. Photomicrographs were captured with an HT7800 transmission electron microscope camera. Scale bar represents 5 μm (magnification, × 2,500), 2 μm (magnification, × 6,000) and 1 μm (magnification, × 15,000). (D) Circular map of R. barbariae str. BIME genome. From inner circle to outer circle representative GC skew, GC content, proteins of – strand, contig, proteins of + strand. The location of tRNA, rRNA, ompA, ompB, gltA, 17-kDa, sca1, sca4 and 16S rRNA genes within the genome are indicated. (E) Phylogenetic tree constructed using the maximum likelihood method with 1,000 replications, based on the whole genomes of 24 other publicly available established or proposed Rickettsiales species. Anaplasma marginale and Ehrlichia muris were used as outgroup species. Scale bar indicates 0.1 nucleotide substitutions per site.

After stable cultivation, Candidatus R. barbariae strain BIME was purified and subjected to next-generation sequencing, generating over 30 million 150-bp reads. The draft genome of Candidatus R. barbariae was composed of five contigs by de novo assembly, with contig sizes ranging from 88,634–424,156 bp, ultimately forming a circular chromosome of 1,243,886 bp (Figure 1D). The ANI of the Candidatus R. barbariae genome was most closely related to Rickettsia parkeri (R. parkeri) and Rickettsia africae (R. africae), with ANI values of 98.81% and 98.79% (Figure S2), respectively. Accordingly, Candidatus R. barbariae strain BIME was a unique rickettsial species and we modified the proposed name to R. barbariae to conform to the International Code of Nomenclature of Prokaryotes [36,37]. Table 1 compares the R. barbariae genome (GenBank accession no. JBEWYL000000000) with other representative strains of SFGR, indicating that R. barbariae had a smaller genome size compared to other SFGRs, with a GC content similar to others at 32.4%. Gene annotation revealed that R. barbariae had 1,461 genes, including 1,425 protein-coding genes, along with three rRNAs and 33 tRNAs. A phylogenetic tree based on the whole genomes indicated that R. barbariae was most closely related to R. parkeri and R. africae (Figure 1E), suggesting potential pathogenic capabilities similar to these species. Table 1. Genomic characteristics of Rickettsia barbariae compared with other representative Rickettsia strains.

Characteristic	Rickettsia barbariae str. BIME	Rickettsia parkeri str. Grand Bay	Rickettsia africae str. ESF-5	Rickettsia sibirica str. 246	Rickettsia slovaca str. 13-B	
Accession	JBEWYL000000000	GCA_000965085.1	GCA_000023005.1	GCA_000166935.1	GCA_000237845.1	
Genome size (bp)	1,243,886	1,309,691	1,290,917	1,250,021	1,275,089	
BUSCO (%)	98.90 (F:0.3, M:0.8)	99.40 (F:0.0, M:0.6)	99.70 (F:0.0, M:0.3)	98.90 (F:0.0, M:1.1)	98.90 (F:0.8, M:0.3)	
GC content (%)	32.42	32.44	32.41	32.47	32.50	
Gene counts	1,461	1,507	1,492	1,460	1,493	
CDS	1,425	1,470	1,452	1,424	1,453	
tRNAs	33	34	33	33	33	
rRNAs	3	3	3	3	3	
No. of contigs	5	1	2	1	1	
N50 (bp)	363,995	1,309,691	1,278,540	1,250,021	1,275,089	
L50	2	1	1	1	1	
N90 (bp)	101,403	1,309,691	1,278,540	1,250,021	1,275,089	
L90	4	1	1	1	1	

Annotation of R. barbariae genome in comparison to other SFGR species

To explore the gene differences between R. barbariae and other SFGRs, the orthogroups were compared with closely related SFGRs (Figure 2A). These SFGRs shared a common set of 1,125 orthogroups, with R. barbariae lacking any unique orthogroups. We then annotated all the genes of R. barbariae, R. parkeri, R. africae, and Rickettsia sibirica using the COG and KEGG databases for understanding the differences in gene function between R. barbariae and closely related SFGRs. Annotation using the COG database (Figure 2B) showed that, similar to other SFGRs, the top three classifications of R. barbariae proteins were Class S (Function unknown), Class J (Translation, ribosomal structure, and biogenesis), and Class M (Cell wall/membrane/envelope biogenesis). Notably, R. barbariae exhibited fewer proteins classified in Class U (Intracellular trafficking, secretion, and vesicular transport) compared to other SFGRs (Table S2), suggesting potential limitations in intracellular transport functions within host cells, which may affect its growth and replication capabilities [38,39]. The KEGG database annotation (Table S3) showed that, like other SFGRs, the top three categories of R. barbariae proteins were genetic information processing, signalling and cellular processes, and metabolism. Similarly, R. barbariae showed fewer proteins in the membrane transport category compared to R. parkeri and R. africae, consistent with the results from the COG database. Figure 2. Functional annotation of R. barbariae str. BIME genome. (A) UpsetR plot showing the number of orthogroups in the genome of R. barbariae compared with other closely related SFGR representatives. Connected circles indicate shared orthogroups among these SFGR species. (B) COG annotation of R. barbariae and other closely related SFGR genomes. (C) Heatmap illustrating the presence of virulence factors in R. barbariae and other representative SFGR genomes. The vertical axis represents various SFGRs, the horizontal axis indicates the names of the virulence factors, the size of the bubbles represents the coverage of the virulence factors, and the colour intensity indicates their identity.

All eight types of virulence factors, which are present in the genomes of pathogenic SFGR, were identified in the R. barbariae str. BIME (Figure 2C) with some differences in coverage and identity. Compared to the closely related R. parkeri, R. barbariae showed variation in the coverage of the pat2 protein, which possesses phospholipase A2 activity. This protein facilitates Rickettsia entry into host cells, escape from phagosomes, and dissolution of host cells [40,41]. Importantly, R. barbariae possesses the rvhB6b gene absent in R. parkeri, which belongs to the VirB6-like protein of the type IV secretion system. This gene is primarily involved in delivering bacterial macromolecules to host cells [42,43], suggesting potentially enhanced virulence of R. barbariae with regard to effector molecule delivery systems.

Phylogenetic analyses

To investigate the prevalence of R. barbariae in ticks, we conducted specific PCRs targeting the ompA (532 bp), ompB (526 bp), gltA (381 bp), sca1 (657 bp), and sca4 (920 bp) genes of SFGR (Table S1) to screen 66 Rh. turanicus ticks collected from Wujiaqu City. Two samples were positive for R. barbariae, resulting in an infection rate of 3.03%. Sanger sequencing confirmed these results, with sequences deposited in GenBank under accession numbers PP954847–PP954858 (Table S4). Phylogenetic analyses based on the ompA, ompB, gltA, sca1, and sca4 genes were conducted to further understand the genetic variation and evolutionary relationships among R. barbariae strains (Figure 3). The sequences of the ompA, ompB, sca1, and sca4 genes amplified from R. barbariae isolates XJ-1 and XJ-2 were identical to those of R. barbariae str. BIME. Phylogenetic analysis based on the gltA gene revealed that R. barbariae isolates XJ-1 and XJ-2 (GenBank accession nos. PP954847 and PP954848) exhibited 99.72% homology with R. barbariae str. BIME, and 100.00% homology with Candidatus R. barbariae sequences from human blood and urine samples reported in China (GenBank accession nos. MW314837, MW321555 and MW321556). The ompB and sca4 gene sequences of R. barbariae isolates XJ-1 and XJ-2 (GenBank accession nos. PP954853–54, PP954857–58) showed 100% homology with those of Candidatus R. barbariae sequences from human (GenBank accession nos. MW314838 and MW321585). Phylogenetic trees based on the ompA gene sequences of R. barbariae isolates XJ-1 and XJ-2 (GenBank accession nos. PP954850 and PP954851) had a similarity range of 99.79–100.00% with Candidatus R. barbariae strains from human (GenBank accession nos. MW314829, MW321563, and OM001343), while the sca1 gene sequences showed 99.64% similarity with isolates from human in Xinjiang, China (GenBank accession nos. MW307786 and MW321580). Figure 3. Phylogenetic analysis of Rickettsia barbariae based on the nucleotide sequences of five genes. (A) Phylogenetic tree based on the ompA gene. (B) Phylogenetic tree based on the ompB gene. (C) Phylogenetic tree based on the gltA gene. (D) Phylogenetic tree based on the sca1 gene. (E) Phylogenetic tree based on the sca4 gene. Bootstrap analysis with 1,000 replicates was conducted to evaluate phylogenetic robustness. Scale bar indicates the number of nucleotide substitutions per site. Sequences highlighted in red font represent those obtained in this study, while those in blue font denote Candidatus R. barbariae sequences identified from human samples in GenBank.

Discussion

This study represents the first isolation of the R. barbariae str. BIME from the eggs of Rh. turanicus ticks in Xinjiang, China. Utilizing next-generation sequencing and assembly, we obtained the whole genome sequence and further characterized its genomic and genetic evolutionary features. Phylogenetic analysis indicates that this emerging Rickettsia is a distinct species and most closely related to pathogenic R. parkeri and R. africae.

Since the first detection in Rh. turanicus in Italy in 2008, Candidatus R. barbariae has been found in various vectors and hosts across multiple countries [4–12]. However, the lack of isolated strains and whole genome sequences has limited the ability to further explore its biological and genomic characteristics. The diversity of microorganisms harboured by ticks collected in the field is extensive, complicating the isolation of pathogens by direct cell culture due to the potential for exogenous contamination. Given that SFGRs are transmitted transovarially [13,14], we chose to isolate pathogens using eggs obtained from engorged female ticks. This method effectively eliminated the interference from microorganisms not transmitted transovarially, thereby enhancing the efficacy of pathogen isolation.

R. barbariae has been detected in ticks and wild animals, demonstrating broad adaptability and transmission capability. In this study, we successfully isolated R. barbariae from the eggs of Rh. turanicus, further confirming that this tick species may be a primary vector for R. barbariae. Rh. turanicus is widely distributed in Xinjiang and is one of the dominant tick species [44,45]. Surveys on SFGR prevalence in tick species in Xinjiang region of China have shown that infection rates of R. barbariae in Rh. turanicus ranging from 21.20% to 30.76% [9,10]. Rh. turanicus primarily infests domestic animals such as sheep, goats, cattle, and dogs [46,47], thereby increasing the risk of human exposure to tick bites and pathogen infection. Notably, sequences available in GenBank show that this emerging Rickettsia has been detected in human whole blood and urine samples from Xinjiang, China. Phylogenetic analysis (Figure 3) indicates that R. barbariae str. BIME and the gene sequences detected in Rh. turanicus ticks are highly consistent with sequences previously found in humans and vector ticks in Xinjiang, further suggesting a high risk of human infection. Additionally, R. barbariae has also been detected in the spleen and liver of the Vormela peregusna [11], indicating the host tissue tropism of this pathogen in natural infection, which also provides clues for our subsequent investigation of its pathogenic mechanisms.

Through next-generation sequencing of purified cultures, we found that the genome size and gene count of R. barbariae exhibit distinct differences compared to other SFGRs, potentially influencing its ability to adapt to an intracellular lifestyle. Although a previous study attempted to sequence the genome of Candidatus R. barbariae from a Rh. turanicus tick in Palestine [48] and preliminarily reported a genome size of 1,246,042 bp, which differs from the 1,243,886 bp obtained in this study, the genome sequence was unfortunately not publicly released. This limits our ability to conduct further comparisons of genome characteristics and to investigate the specific genes responsible for these genome size differences and their potential biological implications.

In comparative genomic analysis, we identified both differences and similarities in genes between R. barbariae and other SFGRs. All virulence factors expressed by SFGRs known to cause human disease are present in R. barbariae. The similarity between these genomes suggests that this emerging Rickettsia is potentially pathogenic to humans. There are also some differences in the genome of R. barbariae compared to that of other SFGRs, and these variations likely reflect distinct strategies employed by different strains to adapt to hosts and environments [49]. For instance, we observed a reduction in the number of genes involved in intracellular and membrane transport functions in R. barbariae, suggesting potential differences in its survival and replication within host cells compared to other SFGRs. Specifically, the coverage of the exotoxin-like pat2 gene in R. barbariae was lower compared to R. parkeri and R. africae, which is implicated in Rickettsia entry into host cells, escape from phagosomes, and damage to host cells [40,41]. Pre-treatment of R. typhi with antibodies targeting the pat2 protein significantly reduced its ability to infect Vero cells compared to the control group in plaque assays. Regarding the effector delivery system, the coverage of the rvhB6b gene was higher in R. barbariae compared to R. africae and R. parkeri, especially since R. parkeri lacks the rvhB6b gene altogether. The rvhB6b gene belongs to the T4SS effector class and its role in SFGR pathogenesis is still under investigation. Studies in Anaplasma phagocytophilum suggest that T4SS secretes virulence factors that subvert innate immunity and inhibit host cell apoptosis [43,50]. Understanding how these virulence genes impact the pathogenic capabilities of R. barbariae compared to other SFGRs warrants further exploration.

A limitation of this study is that it was conducted in a representative area of northwestern China. The geographic distribution and evolutionary characteristics of this Rickettsia in other regions remain unclear, which hinders our ability to comprehensively compare strains from different areas. In addition, although the virulence genes of this Rickettsia, similar to those found in pathogenic SFGRs, have been identified through genomic characterization, its pathogenicity requires further validation through population surveillance in high-risk areas.

In conclusion, this study represents the first isolation and genomic characterization of R. barbariae from the eggs of engorged Rh. turanicus ticks in northwestern China. By filling the existing gaps in genomic data and isolate availability for this emerging Rickettsia, we provide critical insights into its potential pathogenicity, including the identification of all virulence factors present in pathogenic SFGRs within the R. barbariae isolate. Phylogenetic analysis further reveals its close evolutionary relationship with known pathogens R. parkeri and R. africae, highlighting the potential public health threat posed by R. barbariae. These findings not only establish a foundational understanding of the genomic and evolutionary characteristics of R. barbariae but also underscore the significant pathogenic potential that warrants immediate attention. Enhanced surveillance and targeted preventive measures should be prioritized, particularly in high-risk areas, to mitigate the potential health risks associated with this emerging Rickettsia.

Supplementary Material

Appendix.pdf

Disclosure statement

No potential conflict of interest was reported by the author(s).
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References

1 Seidi S, Omidi AH, Esmaeili S. Distribution of different Rickettsia species in countries of the WHO Eastern Mediterranean (WHO-EMRO) region: An overview. Travel Med Infect Dis. 2024;58 :102695. doi:10.1016/j.tmaid.2024.102695 38360158
2 Fang LQ, Liu K, Li XL, et al. Emerging tick-borne infections in mainland China: an increasing public health threat. Lancet Infect Dis. 2015;15 :1467–1479. doi:10.1016/s1473-3099(15)00177-2 26453241
3 Zhang YY, Sun YQ, Chen JJ, et al. Mapping the global distribution of spotted fever group rickettsiae: a systematic review with modelling analysis. Lancet Digit Health. 2023;5 :e5–e15. doi:10.1016/s2589-7500(22)00212-6 36424337
4 Mura A, Masala G, Tola S, et al. First direct detection of rickettsial pathogens and a new rickettsia, ‘Candidatus rickettsia barbariae’, in ticks from Sardinia, Italy. Clin Microbiol Infect. 2008;14 :1028–1033. doi:10.1111/j.1469-0691.2008.02082.x 19040474
5 Cicculli V, Capai L, Quilichini Y, et al. Molecular investigation of tick-borne pathogens in ixodid ticks infesting domestic animals (cattle and sheep) and small rodents (black rats) of Corsica, France. Ticks Tick Borne Dis. 2019;10 :606–613. doi:10.1016/j.ttbdis.2019.02.007 30777731
6 Sukhiashvili R, Zhgenti E, Khmaladze E, et al. Identification and distribution of nine tick-borne spotted fever group rickettsiae in the country of Georgia. Ticks Tick Borne Dis. 2020;11 :101470. doi:10.1016/j.ttbdis.2020.101470 32723640
7 Ji S, Ceylan O, Ma Z, et al. Protozoan and rickettsial pathogens in ticks collected from infested cattle from Turkey. Pathogens. 2022;11 :500. doi:10.3390/pathogens11050500 35631021
8 Perfilyeva YV, Berdygulova ZA, Mashzhan AS, et al. Molecular and seroepidemiological investigation of Сoxiella burnetii and spotted fever group rickettsiae in the southern region of Kazakhstan. Ticks Tick Borne Dis. 2023;14 :102240. doi:10.1016/j.ttbdis.2023.102240
9 Guo LP, Jiang SH, Liu D, et al. Emerging spotted fever group rickettsiae in ticks, northwestern China. Ticks Tick Borne Dis. 2016;7 :1146–1150. doi:10.1016/j.ttbdis.2016.08.006 27554852
10 Song S, Chen C, Yang M, et al. Diversity of Rickettsia species in border regions of northwestern China. Parasit Vectors. 2018;11 :634. doi:10.1186/s13071-018-3233-6 30545379
11 Liu X, Yang M, Liu G, et al. Molecular evidence of Rickettsia raoultii, “Candidatus Rickettsia barbariae” and a novel Babesia genotype in marbled polecats (Vormela peregusna) at the China-Kazakhstan border. Parasit Vectors. 2018;11 :450. doi:10.1186/s13071-018-3033-z 30075738
12 Liu G, Zhao S, Tan W, et al. Rickettsiae in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks in northwestern China. Parasit Vectors. 2021;14 :204. doi:10.1186/s13071-021-04718-1 33874985
13 Socolovschi C, Huynh TP, Davoust B, et al. Transovarial and trans-stadial transmission of Rickettsiae africae in Amblyomma variegatum ticks. Clin Microbiol Infect. 2009;15 (Suppl 2 ):317–318. doi:10.1111/j.1469-0691.2008.02278.x 19456811
14 Guizzo MG, Budachetri K, Adegoke A, et al. Rickettsia parkeri infection modulates the sialome and ovariome of the Gulf coast tick, Amblyomma maculatum. Front Microbiol. 2022;13 :1023980. doi:10.3389/fmicb.2022.1023980 36439862
15 Roux V, Fournier PE, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. Journal Clin Microbiol. 1996;34 :2058–2065. doi:10.1128/jcm.34.9.2058-2065.1996
16 Yuan TT, Du CH, Xia LY, et al. Molecular evidence of Candidatus Rickettsia longicornii and a novel Rickettsia strain from ticks in Southern China. Ticks Tick Borne Dis. 2021;12 :101679. doi:10.1016/j.ttbdis.2021.101679 33578256
17 Zhao SS, Li HY, Yin XP, et al. First detection of Candidatus Rickettsia barbariae in the flea Vermipsylla alakurt from north-western China. Parasit Vectors. 2016;9 :325. doi:10.1186/s13071-016-1614-2 27267467
18 Zhao S, Yang M, Jiang MM, et al. Rickettsia raoultii and Rickettsia sibirica in ticks from the long-tailed ground squirrel near the China-Kazakhstan border. Exp Appl Acarol. 2019;77 :425–433. doi:10.1007/s10493-019-00349-5 30805816
19 Du LF, Zhang MZ, Yuan TT, et al. New insights into the impact of microbiome on horizontal and vertical transmission of a tick-borne pathogen. Microbiome. 2023;11 :50. doi:10.1186/s40168-023-01485-2 36915209
20 Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9 :357–359. doi:10.1038/nmeth.1923 22388286
21 Li H, Handsaker B, Wysoker A, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25 :2078–2079. doi:10.1093/bioinformatics/btp352 19505943
22 Bankevich A, Nurk S, Antipov D, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19 :455–477. doi:10.1089/cmb.2012.0021 22506599
23 Kang DD, Li F, Kirton E, et al. MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ. 2019;7 :e7359. doi:10.7717/peerj.7359 31388474
24 Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;3 :2068–2069. doi:10.1093/bioinformatics/btu153
25 Parks DH, Imelfort M, Skennerton CT, et al. Checkm: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25 :1043–1055. http://www.genome.org/cgi/doi/10.1101gr.186072.114.25977477
26 Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90 K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9 :5114. doi:10.1038/s41467-018-07641-9 30504855
27 Asnicar F, Thomas AM, Beghini F, et al. Precise phylogenetic analysis of microbial isolates and genomes from metagenomes using PhyloPhlAn 3.0. Nat Commun. 2020;11 (1 ):2500. doi:10.1038/s41467-020-16366-7 32427907
28 Minh BQ, Schmidt HA, Chernomor O, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37 :1530–1534. doi:10.1093/molbev/msaa015 32011700
29 McGinnis S, Madden TL. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 2004;3 :W20–W25. doi:10.1093/nar/gkh435
30 Rozewicki J, Li S, Amada KM, et al. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Res. 2019;47 :W5–W10. doi:10.1093/nar/gkz342 31062021
31 Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. Trimal: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;2 :1972–1973. doi:10.1093/bioinformatics/btp348
32 Yu G, Lam TTY, Zhu H, et al. Two methods for mapping and visualizing associated data on phylogeny using ggtree. Mol Biol Evol. 2018;35 :3041–3043. doi:10.1093/molbev/msy194 30351396
33 Emms DM, Kelly S. Orthofinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20 :238. doi:10.1186/s13059-019-1832-y 31727128
34 Conway JR, Lex A, Gehlenborg N. Upsetr: an R package for the visualization of intersecting sets and their properties. Bioinformatics. 2017;33 :2938–2940. doi:10.1093/bioinformatics/btx364 28645171
35 Cantalapiedra CP, Hernández-Plaza A, Letunic I, et al. EggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol. 2021;38 :5825–5829. doi:10.1093/molbev/msab293 34597405
36 Oren A, Vandamme P, Schink B. Notes on the use of Greek word roots in genus and species names of prokaryotes. Int J Syst Evol Microbiol. 2016;66 :2129–2140. doi:10.1099/ijsem.0.001063 27055242
37 Karpathy SE, Slater KS, Goldsmith CS, et al. Rickettsia amblyommatis sp. nov., a spotted fever group Rickettsia associated with multiple species of Amblyomma ticks in North, Central and South America. Int J Syst Evol Microbiol. 2016;66 :5236–5243. doi:10.1099/ijsem.0.001502 27638476
38 Fitzsimmons L, Bublitz D, Clark T, et al. Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN-β signaling in primary human dermal microvascular endothelial cells. mBio. 2024;15 :e0345023. doi:10.1128/mbio.03450-23 38445878
39 Huang D, Luo J, OuYang X, et al. Subversion of host cell signaling: the arsenal of Rickettsial species. Front Cell Infect Microbiol. 2022;12 :995933. doi:10.3389/fcimb.2022.995933 36389139
40 Borgo GM, Burke TP, Tran CJ, et al. A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes. Nat Commun. 2022;13 :3656. doi:10.1038/s41467-022-31351-y 35760786
41 Rahman MS, Gillespie JJ, Kaur SJ, et al. Rickettsia typhi possesses phospholipase A2 enzymes that are involved in infection of host cells. PLoS Pathog. 2013;9 :e1003399. doi:10.1371/journal.ppat.1003399 23818842
42 Bao W, Kumagai Y, Niu H, et al. Four VirB6 paralogs and VirB9 are expressed and interact in Ehrlichia chaffeensis-containing vacuoles. J Bacteriol. 2009;191 :278–286. doi:10.1128/jb.01031-08 18952796
43 Al-Khedery B, Lundgren AM, Stuen S, et al. Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum. BMC Genomics. 2012;13 :678. doi:10.1186/1471-2164-13-678 23190684
44 Ma R, Li C, Gao A, et al. Tick species diversity and potential distribution alternation of dominant ticks under different climate scenarios in Xinjiang, China. PLoS Negl Trop Dis. 2024;18 :e0012108. doi:10.1371/journal.pntd.0012108 38683839
45 Sheng J, Jiang M, Yang M, et al. Tick distribution in border regions of Northwestern China. Ticks Tick Borne Dis. 2019;10 :665–669. doi:10.1016/j.ttbdis.2019.02.011 30833199
46 Ma Z, Ceylan O, Galon EM, et al. Molecular identification of piroplasmids in ticks from infested small ruminants in Konya Province, Turkey. Pathogens. 2023;12 :1123. doi:10.3390/pathogens12091123 37764931
47 Zeb J, Song B, Senbill H, et al. Ticks infesting dogs in Khyber Pakhtunkhwa, Pakistan: detailed epidemiological and molecular report. Pathogens. 2023;12 :98. doi:10.3390/pathogens12010098 36678446
48 Ravi A, Ereqat S, Al-Jawabreh A, et al. Metagenomic profiling of ticks: identification of novel rickettsial genomes and detection of tick-borne canine parvovirus. PLoS Negl Trop Dis. 2019;13 :e0006805. doi:10.1371/journal.pntd.0006805 30640905
49 Merhej V, Raoult D. Rickettsial evolution in the light of comparative genomics. Biol Rev Camb Philos Soc. 2011;86 :379–405. doi:10.1111/j.1469-185X.2010.00151.x 20716256
50 Rikihisa Y, Lin M, Niu H. Type IV secretion in the obligatory intracellular bacterium Anaplasma phagocytophilum. Cell Microbiol. 2010;12 :1213–1221. doi:10.1111/j.1462-5822.2010.01500.x 20670295
