High diversity of Rickettsia spp., Anaplasma spp., and Ehrlichia spp. in ticks from Yunnan Province, Southwest China

Rickettsia, Anaplasma, and Ehrlichia belonging to the order Rickettsiales are causative agents of tick-borne diseases in humans. During 2021, 434 ticks including Rhipicephalus microplus and R. haemaphysaloides were collected from three sampling sites in Yunnan Province, Southwest China, and analyzed for the presence of these bacteria. Nine bacterial species were identified, including two Rickettsia spp., three Anaplasma spp., and four Ehrlichia spp., some of which are potential human pathogens. Genetic and phylogenetic analysis on 16S rRNA, gltA, groEL, ompA, ompB, and sca4 genes indicated the presence of a novel spotted fever group Rickettsia (SFGR) named “Candidatus Rickettsia shennongii” in six of the 38 R. haemaphysaloides ticks from two locations, Dehong Autonomous Prefecture and Honghe City. Another SFGR species, Candidatus Rickettsia jingxinensis was detected in ticks from all three sites, with an overall positive rate of 62.67%. Three other human pathogenic species, Anaplasma ovis (1.38%, 6/434), Ehrlichia canis (16.36%, 71/434), and E. chaffeensis (0.23%, 1/434) were detected in these ticks and characterized. Moreover, Ehrlichia sp. (4.84%, 21/434), E. minasensis (7.37%, 32/434), A. marginale (6.91%, 30/434), and Cadidatus Anaplasma boleense (1.15%, 5/434) were detected in R. microplus ticks, for which pathogenicity to humans remains to be determined. The results reveal the remarkable diversity of Rickettsiales bacteria in ticks from Yunnan Province, Southwest China. The high infection rate of some human pathogenic bacteria in ticks may indicate potential infection risk in humans, and it highlights the need for surveillance in local populations.


Introduction
in Rhipicephalus microplus ticks from Yunnan, including Ca. R. jingxinensis, A. marginale, and Coxiella burnetii. Furthermore, A. capra, A. phagocytophilum, and Candidatus Neoehrlichia mikurensis were also detected in R. microplus ticks from Yunnan Province (Jiao et al., 2021). Although many studies on tick-borne Rickettsiales bacteria have been performed in Yunnan Province, further exploration is still needed. To improve our knowledge on the biodiversity and epidemiology of Rickettsiales bacteria, we collected ticks from goats and cattle in three locations of Yunnan Province, and characterized Rickettsia, Anaplasma, and Ehrlichia in them.

Sample collection and processing
During 2021, ticks were collected from three locations in Yunnan Province: Ruili county-level city of Dehong Dai-Jingpo Autonomous Prefecture (97.85°E, 24.01°N), Zhaoyang District of Zhaotong city (103.71°E, 24.32°N), and Shiping county of Honghe city (102.49°O, 23.71°N; Figure 1). Ticks were collected from cattle and goats, then brought to China CDC alive. Tick species were morphologically characterized by a sophisticated arthropod taxonomist based on the characteristics of the capitula, body, legs, anal groove, and caudal appendage (Namgyal et al., 2021). For further confirmation, randomly selected ticks were undergone molecular analysis by sequencing the mitochondrial cytochrome oxidase I (COI) gene sequences (Lu et al., 2013).

DNA extraction, PCR detection, and amplification of key genes
After washing three times with sterile phosphate-buffered saline (PBS), ticks were individually homogenized in 500 μl of PBS Each tick DNA sample was screened by PCR for the presence of bacterial DNA including Rickettsia spp., Anaplasma spp., and Ehrlichia spp. Rickettsial DNA was detected by nested PCR as described previously using primers targeting the outer membrane protein A (ompA) gene, resulting in amplification of a 755-bp fragment (Lu et al., 2017). The DNA samples were also screened for Anaplasma and Ehrlichia DNA as described previously, amplifying a 550-850 bp fragment of the 16S rRNA gene (Guo et al., 2016). Negative control with distilled water in the PCR master mixture and positive control (DNA of R. felis, A. marginale and E. canis, respectively) were included in each test. After 1% agarose gel electrophoresis, amplification products were subjected to sequencing.
For further characterization and phylogenetic analysis of the detected bacterial strains, the partial sequences of the gltA gene (citrate synthase), the groEL gene (heat shock protein), and a long fragment of the 16S rRNA gene were obtained for representative Rickettsia, Anaplasma, and Ehrlichia positive samples. Additionally, nearly complete sequences of ompB and sca4 genes were obtained for the putative novel Rickettsia species. The primers used were described previously (Roux and Raoult, 2000;Sekeyova et al., 2001;Kang et al., 2014;Guo et al., 2016;Lu et al., 2017) and they are listed in Supplementary Table S1. All recovered sequences have been deposited in the GenBank database (GenBank numbers listed in Supplementary Table S2).

Genetic and phylogenetic analysis of obtained sequences
Sequences obtained in this study were analyzed by BLASTn 1 and Clustal W within Molecular Evolutionary Genetics Analysis (MEGA) software, version 7.0 (Kumar et al., 2016). Phylogenetic analysis was also performed using PhyML 3.0. Confidence values for each branch of the phylogenetic trees were determined by bootstrap analysis with 1,000 replicates. Sequences recovered in this study were aligned with the reference sequences retrieved from the GenBank database.

Sample collection
During March to July 2021, 434 ticks (397 R. microplus and 37 R. haemaphysaloides) were collected from three locations in Yunnan Province. The three sampling locations (Ruili County-level City of Dehong Dai-Jingpo Autonomous Prefecture, Zhaoyang District of Zhaotong City, and Shiping County of Honghe City) were shown in Figure 1. Tick species were initially determined by morphological examination and then further determined by amplification and analysis of the COI gene (Lu et al., 2013). Except for four nymphs from Zhaotong, all ticks were adult, and most were fully or partially engorged. The COI sequences of ticks have been submitted to GenBank. The Accession numbers are OM959242-OM959313, OM959315-OM959321, and OM977035-OM977043. The tick species, quantity, and vertebrate hosts in different sampling sites were shown in Supplementary Table S3.

Rickettsia spp.
Nested PCR targeting the conserved domain of the ompA gene was performed to screen for Rickettsia in the collected tick samples. Based on agarose gel electrophoresis, DNA sequencing, and comparison with BLASTn, two Rickettsia species were initially identified: Ca. R. jingxinensis and a putative novel species. For further characterization, the 16S rRNA gene (1,172 bp), as well as the gltA (1,004 bp) and groEL (1038-1,042 bp) genes were successfully amplified from the total tick DNA samples.
DNA of Ca. R. jingxinensis was detected in ticks from all three sites, with strikingly high positive rates varying from 37.50 to 84.69% (48 of 128 ticks from Dehong, 166 of 196 ticks from Zhaotong, and 70 of 110 ticks from Honghe; Table 1). All Ca. R. jingxinensis strains were detected in R. microplus ticks except for one from R. haemaphysaloides. All the ompA sequences shared 100% identity with Ca. R. jingxinensis isolate Meixian-Hl-107 (MH932061.1) and Xian-Hl-79 (MH932069.1) reported in Haemaphysalis longicornis from ShaanXi Province, Northern China (Guo et al., 2019). The 16S rRNA, gltA and groEL genes shared 99.90-100% identity with each other in reference to homologous genes. The 16S rRNA and gltA gene sequences were 100 and 99.90% identical to other Ca. R. jingxinensis strains from China. Meanwhile, the groEL sequences shared highest 99.90% identity with Uncultured Rickettsia sp. clone tick28 (ON409665) and Uncultured Rickettsia sp. clone tick26 (ON409664) we previously identified in Ngawa, Sichuan Province, which actually also represent Ca. R. jingxinensis strains.
Three Anaplasma species were identified; Candidatus Anaplasma boleense, A. ovis, and A. marginale. DNA of Ca.

Ehrlichia spp.
Of the 434 ticks screened from three locations, 125 (28.80%) were positive for Ehrlichia including four species: E. minasensis, E. canis, E. chaffeensis, and Ehrlichia sp. (Table 1). In R. microplus ticks from both Dehong and Honghe city, an Ehrlichia sp. species closely related to Ehrlichia sp. strain WHBMXZ-43 was identified, which was first identified in R. microplus ticks from Wuhan city, South-Central China. The 16S rRNA genes of the detected strains share 99.84-99.91% identity with this Ehrlichia, while both gltA and groEL genes share 100% identity with this strain.
In R. microplus ticks from Honghe city, E. minasensis was detected, with a positive rate of 31.07% (32/103). The 16S rRNA,  (Figure 4). Additionally, an E. chaffeensis strain was detected in a single R. microplus tick. All 16S rRNA, gltA, and groEL genes share 100% similarity with E. chaffeensis str. West Paces, E. chaffeensis Arkansas, and other previously reported E. chaffeensis strains. These appear to be the only gltA and groEL sequences of E. chaffeensis from China submitted to GenBank.

Discussion
Yunnan Province is a widely recognized biodiversity hotspot in China and worldwide . In the present study, we revealed the extensive diversity and high positive rate of Rickettsiales bacteria in ticks from Yunnan Province. Nine   . It has since been detected in ticks from multiple provinces in China, including Shaanxi, Guangxi, Sichuan, and Yunnan (Guo et al., 2019;Liu et al., 2020;Jiao et al., 2021;Lu et al., 2022). Furthermore, it was also reported in Korea, Thailand, and India, spanning from East Asia to South Asia (Takhampunya et al., 2019;Bang et al., 2021) (GenBank No. MN463681-MN463688). Previous studies revealed strikingly high positive rates of this Rickettsia in certain areas (for instance, 44.4-69.7% in H. longicornis from Shaanxi and 24.61%in R. microplus from Yunnan; Guo et al., 2019;Jiao et al., 2021). Our findings confirmed the widespread circulation of Ca. R. jingxinensis in China, as well as its high positive rate in ticks removed from livestock. Because most ticks were fully or partially engorged in the present work, it is not clear whether the Rickettsia DNA was from the blood meal or the tick itself. In another word, the possibility remains that the livestock in these locations may be infected by Ca. R. jingxinensis. These results remind us that the potential risk of Ca. R. jingxinensis infecting animals should be considered, and more attention should be paid to its role in human/animal diseases.
In R. haemaphysaloides ticks from Dehong and Honghe, a novel SFGR species named Ca. R. shennongii was identified. According to the gene sequence-based criteria for the identification of a new Rickettsia species (Fournier et al., 2003), genetic analysis of key genes clearly indicated that these strains represent a novel Rickettsia species. Sequences from all strains were almost identical, and they formed distinct clades in the phylogenetic trees. Genetically, this Rickettsia is closely related to R. raoultii, R. massiliae, and R. conorii (16S rRNA gene shares 99.91% identity with R. raoultii and R. conorii, while groEL gene shares 99.33% identity with R. massiliae), all of which are recognized human pathogens causing spotted fever. Therefore, the pathogenicity of this Rickettsia is a concern. Interestingly, this Rickettsia was only detected in R. haemaphysaloides, a widely distributed three-host tick in China and South Asian countries (Zhou et al., 2006). This species is known to harbor pathogens including A. phagocytophilum, A. ovis, R. rhipicephali, R. slovaca, R. massiliae, and Babesia microti (Kuo et al., 2018;Li et al., 2018;Ghafar et al., 2020;Ali et al., 2021). Our results may suggest the possibility of R. haemaphysaloides ticks harboring Ca. R. shennongii.  Herein, the DNA of three Anaplasma species was detected in all three locations including Candidatus Anaplasma boleense, A. ovis, and A. marginale. Anaplasma ovis is a widely-distributed pathogen affecting sheep, goats, and wild ruminants. As the etiological agent of ovine anaplasmosis first reported in 1956, the pathogenicity of A. ovis has been well studied since. Anaplasma ovis infection in sheep is usually subclinical and mostly manifested as hemolytic anemia (Dahmani et al., 2019). Other main clinical manifestations include extreme weakness, anorexia, and weight loss, but these manifestations mostly occur under poor health conditions (Bauer et al., 2021). In 2010, a human anaplasmosis case caused by an A. ovis variant was reported in Cyprus, with clinical symptoms of fever, hepatosplenomegaly, and enlarged lymph nodes (Chochlakis et al., 2010), providing evidence that A. ovis might be a potential zoonotic pathogen that may occasionally infect humans. In the present study, ticks from Dehong and Honghe were both tested positive for A. ovis, suggesting that A. ovis circulation is common in Yunnan Province, and surveillance in local populations is needed.
In this study, Ca. A. boleense was detected in ticks from all three sites of Yunnan Province. Candidatus Anaplasma boleense was first identified in ticks from Bole City in the Xinjiang Uygur Autonomous Region, Northwest China (Kang et al., 2014). In recent years, it has been detected in mosquitoes and rodents (FJ182047) in multiple provinces of China (Guo et al., 2016). In 2018, Ca. A. boleense was found to infect deer, boars, buffalos, cows, and bats in Peninsular Malaysia (Koh et al., 2018). In 2020, Ca. A. boleense was reported to infect marsh deer in Argentina, South America (Orozco et al., 2020). Furthermore, some sequences submitted to the GenBank database suggest that this Anaplasma exists in Australia (MH500004) and South Africa (MK814450), suggesting that it is distributed worldwide. However, few studies have been performed on the pathogenicity of Ca. A. boleense toward humans or other animals. The wide host range, as well as its wide geographical distribution, suggest that this Anaplasma species merits further investigation.
Ehrlichia is a genus closely related to human diseases. In this study, the DNA of 4 Ehrlichia species were identified, including E. canis, E. chaffeensis, E. minasensis, and Ehrlichia sp., of which E. canis and E. chaffeensis are recognized human pathogens (Perez et al., 1996;Bouza-Mora et al., 2017). The most prevalent species was E. canis (36.22% positive rate in ticks from Zhaotong), the causative agent of canine monocytic ehrlichiosis. In China, E. canis has been detected in various hosts including ticks, goats, dogs, and deer in multiple locations (Xu et al., 2015;Qiu et al., 2016;Yu et al., 2016;Lu et al., 2017;Zhang et al., 2017). Notably, E. canis is considered a potential agent of human disease (Perez et al., 1996;Bouza-Mora et al., 2017), although human infection cases have never been reported in China. As shown in Figure 2, the strains we detected are most closely related to E. canis strain WHBMXZ-124, a strain identified in R. microplus from Wuhan, China. However, considerable genetic distance exists between them, suggesting that they might represent a variant circulating in China. As a recently recognized species closely related to E. canis, E. minasensis is believed to have evolved from highly variable strains of E. canis, and it has been discovered in Canada, Brazil, France, Pakistan, Ethiopia, etc. . Other than ticks, it also infects mammals including cattle and cervid. In China, there is only one report of E. minasensis in Hainan Province, South China . In our current study, E. minasensis was detected in R. microplus ticks from Honghe city, with a high positive rate of 31.07%. To the best of our knowledge, this is only the second report of E. minasensis circulating in China. Due to its pathogenicity to mammals, the close relationship to human pathogenic species E. canis, and the high infection rate in ticks, its pathogenicity to humans and animals should be further explored.
In summary, this study revealed substantial diversity of Rickettsia, Ehrlichia, and Anaplasma in ticks from Yunnan Province, including a novel Rickettsia species. Notably, some of these bacteria are human pathogens (E. canis, E. chaffeensis, A. ovis, etc) and the positive rates are high. Considering the frequent contact between humans and tick hosts (goats and cattle), these results indicate the potential risk of zoonosis transmitted from ticks to humans. Additionally, the human pathogenicity of Ca. R. shennongii should be further studied and surveillance in these areas is clearly needed.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethics statement
This study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention.

Author contributions
KL conceived the study. KL and ML designed the experiments. KL, JT, and JH collected the samples. ML and WW performed the experiments. HZ, KL, and WG performed data analysis. HZ, HJ, and KL wrote the manuscript. All authors contributed to the article and approved the submitted version.