Prevalence of Spotted Fever Group Rickettsia and Candidatus Lariskella in Multiple Tick Species from Guizhou Province, China

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. “Candidatus Rickettsia jingxinensis” was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63–74.89% and 73.31%) similarities to “Candidatus Midichloria mitochondrii”, respectively. Herein, we name the species “Candidatus Lariskella guizhouensis”. These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.


Introduction
Ticks are one of the most important vectors for pathogens of both humans and animals worldwide [1]. Over the past few decades, more than one hundred tick-borne pathogens, such as spotted fever group Rickettsia, tick-borne encephalitis virus (TBEV), and severe fever with thrombocytopenia syndrome virus (SFTSV), have been identified. In recent years, novel tick-borne pathogens have continued to be reported [2,3]. In 2015, a novel Anaplasma species named Anaplasma capra was detected in 28 patients with symptoms of fever, headache, and malaise in Northeast China [4]. In 2021, a new tick-borne orthonairovirus named Songling virus (SGLV) was identified in patients from Heilongjiang Province, resulting in the infection of 42 hospitalized patients, with headache, fever, fatigue, and dizziness as the main clinical manifestations [5]. In 2021, Kodama  febrile, thrombocytopenia, and leukopenia [6]. As recently as in 2022, serosurveillance in Japan indicated that a novel tick-borne thogotovirus named Oz virus may naturally infect humans and some other mammals [7]. With increased sampling and development in detection techniques, more novel tick-borne human pathogens are still being discovered.
Due to the vast territory and different climates across China, ticks and tick-borne diseases are prevalent in most areas of this country and pose a significant threat to public health [8]. At least 103 tick-borne agents have been detected, 65 of which were identified in the past two decades [8]. However, most studies have focused on common and easily available tick species, such as Rhipicephalus microplus, Haemaphysalis longicornis, and Ixodes persulcatus. Most of the currently known tick-borne agents were reported in these common tick species [8]. Hence, the pathogens harboured by many tick species may have long been underestimated and largely unexplored due to biased sampling.
The order Rickettsiales includes a group of well-recognized human pathogens such as Rickettsia spp., Orientia spp., Ehrlichia spp., Anaplasma spp. and Neoehrlichia spp., most of which are tick-borne pathogens [9]. As notorious human pathogens causing a series of symptoms from fever to even death, Rickettsia, Orientia, Ehrlichia, Neoehrlichia, and Anaplasma, have drawn attention worldwide and been well-studied in many countries and regions [8,10,11]. With the development of high throughput sequencing, some novel families and genera within the order Rickettsiales have been characterized and defined. In 2006, an endosymbiont of the Ixodes ricinus named "Candidatus Midichloria mitochondrii" was described, which also represents a novel genus Candidatus Midichloria within the order Rickettsiales [12]. In 2013, a novel family named "Candidatus Midichloriaceae" was proposed, including genera Candidatus Midichloria, Candidatus Lariskella, Candidatus Nicolleia, Candidatus Fokinia, and Candidatus Aquarickettsia [13]. Although some have been reported to infect humans and mammals such as dogs and horses [14,15], studies on the prevalence, genetic diversity, and pathogenicity of these bacteria are very rare.
To improve the current knowledge on the distribution of ticks and tick-borne Rickettsiales pathogens, ticks collected from Qiandongnan Miao-and-Dong Autonomous Prefecture of Guizhou Province, Southwest China, were thoroughly investigated.

Sample Collection and DNA Extraction
From November to October 2021, ticks were collected in Cengong County (108.82 • O, 27.18 • N), Qiandongnan Miao-and-Dong Autonomous Prefecture, located in Guizhou Province, Southwest China. All the ticks were removed from the body surface of goats and dogs using tweezers, and then stored in 75% alcohol. Morphological identification, mainly based on the characteristic of the anal groove, was carried out by an experienced acarologist to determine the tick species initially. To confirm these results, molecular identification was performed by PCR amplifying and sequencing the partial mitochondrial cytochrome oxidase I (COI) gene of randomly selected tick samples, as described previously [14]. After being washed with Phosphate Buffered Saline (PBS) and thoroughly ground, each tick was subjected to DNA extraction individually using a Mollusc DNA extraction kit (Omega, Norcross, GA, USA) following the instructions. The eluted DNA (60 µL) was stored in a −80 • C refrigerator until molecular identification and PCR detection.

Molecular Detection of Rickettsiales Bacteria
The extracted DNA was screened by PCR analysis of a conserved region of rrs gene to confirm that the bacteria belong to the order Rickettsiales. PCR was performed using Sensoquest PCR System LabCycler (Sensoquest, Göttingen, Germany). As previously shown, PCR amplification screening Rickettsia generates approximately 900 bp products, whereas PCR detecting Anaplasmataceae bacteria generates approximately 450 bp products [16]. The DNA of R. japonica and A. marginale were used as positive controls, whereas ddH 2 O was set as the negative control. After electrophoresis, all the PCR products that met the Biomolecules 2022, 12, 1701 3 of 9 expected size were subjected to DNA sequencing. The recovered rrs sequences were then aligned to those in the GenBank Database to preliminarily determine their genus or species.
The obtained sequences were aligned with the reference sequences in the GenBank Database with BLASTn algorithm to determine their identities to the reported strains. To perform the phylogenetic analysis, the nucleotide sequences of Rickettsia isolates were first manually aligned with those downloaded from the GenBank using the ClustalW method in the MEGA software v3.0. For the Candidatus Lariskella isolates, the rrs sequences were aligned with other Candidatus Lariskella isolates and some Candidatus Midichloriaceae members, whereas the gltA and groEL sequences were only aligned with other Candidatus Midichloriaceae members and some Rickettsiaeae members due to the unavailability of Candidatus Lariskella sequences in GenBank. Phylogenetic trees were constructed based on the maximum likelihood (ML) method by PhyML v3.2 in the GTR+I+G model [18]. The confidence values for each branch were determined by bootstrap analysis with 100 repetitions. All the trees were mid-point rooted and only bootstrap values >70% are shown.
All the sequences in this study have been uploaded to the GenBank Database (shown in Table S2).

Sample Collection and Tick Identification
From November to October 2021, a total of 113 ticks were collected (112 from goats and 1 from a dog) in Cengong County, Qiandongnan Prefecture. Morphological and molecular identification confirmed the existence of 6 tick species: 1 Rhipicephalus microplus, 1 Haemaphysalis longicornis, 3 Haemaphysalis flava, 3 Haemaphysalis kitaokai, 4 Ixodes ovatus, and 101 Ixodes sinensis. BLASTn shows that all the obtained COI sequences (GenBank Accession Numbers: OP107272-OP107273, OP107278-OP107354) have higher than 99% identities to reference COI sequences in the GenBank except for the I. ovatus ticks which have lower identities of approximately 96%. This is consistent with previous reports that I. ovatus contain different phylogenetic groups with remarkable intergroup genetic distances [19]. To our knowledge, this is the first report that H. flava, H. kitaokai, I. ovatus, and I. sinensis are present in Guizhou Province. Phylogenetic analysis results are consistent with the BLASTn results (Figure 1), indicating the remarkable diversity of tick species in this area.
For further study of the detected Candidatus Lariskella isolates, we tentatively designed primers to amplify the gltA and groEL genes based on sequences of other genera of the family Candidatus Midichloriaceae. Subsequently, both the gltA (410-453 bp) and groEL (651 bp) were successfully recovered from four randomly selected isolates. BLASTn alignment showed that the two gene sequences were mostly related to "Candidatus Midichloria mitochondrii". The gltA sequences have 74.63-74.89% identity to "Candidatus Midichloria mitochondrii" IricVA (CP002130), and 74.63-74.71% identity to "Candidatus Nicolleia massiliensis" (DQ788563), both of which belong to the family Candidatus Midichloriaceae, the order Rickettsiales. Meanwhile, the groEL sequences showed 73.31% identity to "Candidatus Midichloria mitochondrii" IricVA (CP002130), and 71.21% to Rickettsiales bacterium Ac37b (CP009217). To the best of our knowledge, these may be the first obtained gltA and groEL sequences of the genus Candidatus Lariskella. In the phylogenetic tree based on the gltA and groEL genes, all four isolates were surrounded by other Candidatus Midichloriaceae members as well as several Rickettsiaceae members, but they were distinct from any other bacterial species and formed an independent clade ( Figure 3). All of these results indicated the representation of an unrecognized species. Herein, we name it "Candidatus Lariskella guizhouensis".
For further study of the detected Candidatus Lariskella isolates, we tentatively designed primers to amplify the gltA and groEL genes based on sequences of other genera of the family Candidatus Midichloriaceae. Subsequently, both the gltA (410-453 bp) and groEL (651 bp) were successfully recovered from four randomly selected isolates. BLASTn alignment showed that the two gene sequences were mostly related to "Candidatus Midichloria mitochondrii". The gltA sequences have 74.63-74.89% identity to "Candidatus Midichloria mitochondrii" IricVA (CP002130), and 74.63-74.71% identity to "Candidatus Nicolleia massiliensis" (DQ788563), both of which belong to the family Candidatus Midichloriaceae, the order Rickettsiales. Meanwhile, the groEL sequences showed 73.31% identity to "Candidatus Midichloria mitochondrii" IricVA (CP002130), and 71.21% to Rickettsiales bacterium Ac37b (CP009217). To the best of our knowledge, these may be the first obtained gltA and groEL sequences of the genus Candidatus Lariskella. In the phylogenetic tree based on the gltA and groEL genes, all four isolates were surrounded by other Candidatus Midichloriaceae members as well as several Rickettsiaceae members, but they were distinct from any other bacterial species and formed an independent clade ( Figure 3). All of these results indicated the representation of an unrecognized species. Herein, we name it "Candidatus Lariskella guizhouensis".

Discussions
To date, approximately 124 tick species belonging to 11 genera have been recorded in China [8]. Although the geographic distribution of various tick species has been well studied, some neglected areas are still largely unexplored. Herein, we collected 113 ticks in Guizhou Province, Southwest China, and molecularly validated the existence of six tick species in them. Of those, I. sinensis, H. flava, H. kitaokai, and I. ovatus had never been recorded in this province. These results may contribute to our knowledge of the geographic distribution of ticks and tick-borne pathogens in China.
Two Rickettsia species were detected in tick samples: Rickettsia monacensis and "Candidatus Rickettsia jingxinensis". As a widely distributed spotted fever group Rickettsia, "Candidatus Rickettsia jingxinensis" has been detected in R. microplus and H. longicornis

Discussions
To date, approximately 124 tick species belonging to 11 genera have been recorded in China [8]. Although the geographic distribution of various tick species has been well studied, some neglected areas are still largely unexplored. Herein, we collected 113 ticks in Guizhou Province, Southwest China, and molecularly validated the existence of six tick species in them. Of those, I. sinensis, H. flava, H. kitaokai, and I. ovatus had never been recorded in this province. These results may contribute to our knowledge of the geographic distribution of ticks and tick-borne pathogens in China.
Two Rickettsia species were detected in tick samples: Rickettsia monacensis and "Candidatus Rickettsia jingxinensis". As a widely distributed spotted fever group Rickettsia, "Candidatus Rickettsia jingxinensis" has been detected in R. microplus and H. longicornis from multiple provinces in China (Liaoning, Guangxi, Sichuan, Hebei, Shaanxi, and Yunnan) and other Asian countries (Korea and India) [16,17,[20][21][22]. Our results have demonstrated the circulation of "Candidatus Rickettsia jingxinensis" in Guizhou Province. Due to its wide geographic distribution and host range, the potential risk to the public health of this Rickettsia should be evaluated. In this study, R. monacensis was detected in three tick species with a total prevalence of 68.14%. Due to the fact that all the ticks were removed from domestic animals, the possibility remains that the detected pathogens are from the blood of infected hosts the ticks fed on. Rickettsia monacensis was first identified in Ixodes ricinus ticks from Germany [23] and has also been detected in other tick species such as Ixodes boliviensis, I. sinensis, Rhipicephalus sanguineus, Hyalomma impeltatum [24][25][26], as well as some mammal hosts such as camels and bats [26,27]. As an SFGR member, R. monacensis was recognized as a human pathogen causing Mediterranean Spotted Fever-like rickettsioses [25]. Although R. monacensis has been detected in several provinces of China [25,28,29], no human infection cases were reported until now. The strikingly high prevalence of R. monacensis in ticks (especially in I. sinensis) may indicate the risk of human infection, especially in populations who are frequently in contact with domestic animals. Furthermore, genetic and phylogenetic analysis indicated that two variants/sub-genotypes of the R. monacensis isolates may have been generated, indicating its long-term evolution in this area. The pathogenicity of the variants/sub-genotypes may warrant further studies.
First proposed in 2012, the genus Candidatus Lariskella has been scarcely studied [30], and little is known about its genetic features. Even with respect to the first identified and most studied species, "Candidatus Lariskella arthropodarum" (previously named Montezuma) [14], only some rrs sequences were available in the GenBank Database until now. Notably, "Candidatus Lariskella arthropodarum" has been detected in acutely febrile patients with the bites of Ixodes ticks in the Far East of Russia [14], suggesting its potential role as a tick-borne human pathogen. In this study, we detected a Candidatus Lariskella species in Ixodes ticks from Guizhou Province, and successfully obtained the gltA and groEL sequences. To our knowledge, these are the first reported gltA and groEL sequences of the genus Candidatus Lariskella. The remarkable genetic distance between this species and other Candidatus Lariskella species indicates that it represents a novel species. Herein we named it "Candidatus Lariskella guizhouensis". It is of interest whether "Candidatus Lariskella guizhouensis" can be transmitted to humans and animals through tick bites.

Conclusions
In conclusion, a high prevalence of Rickettsia monacensis was observed in ticks from Guizhou Province, and they represent at least two variants/sub-genotypes. For this reason, the risk of human infection among local people should be evaluated. In addition, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected and characterized. To the best of our knowledge, this is the first report of gltA and groEL sequences of Candidatus Lariskella. Genetic and phylogenetic analysis indicates that it represents a novel species: "Candidatus Lariskella guizhouensis". This study may contribute to our knowledge of the extensive genetic diversity of Rickettsiales bacteria, and highlights that their potential threat to human health should be further investigated.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom12111701/s1, Table S1. The primers used for amplification of gltA and groEL genes from Candidatus Lariskella guizhouensis by hemi-nested PCR. Table S2. Genbank numbers of the rrs, gltA, groEL, and ompA sequences of Rickettsia and "Candidatus Lariskella guizhouensis" in this study.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequence files are available from the NCBI database (Accession Numbers shown in Table S2).