Pathogenic Rickettsia, Anaplasma, and Ehrlichia in Rhipicephalus microplus ticks collected from cattle and laboratory hatched tick larvae

Background The order Rickettsiales contains a group of vector-borne gram-negative obligate intracellular bacteria, which often cause human emerging infectious diseases and economic losses for dairy and meat industries. The purpose of this study is to investigate the distribution of the pathogens including Rickettsia spp., Anaplasma spp., and Ehrlichia spp. in the order Rickettsiales in ticks from Yueyang, a prefecture-level city of Hunan Province in Sothern China, and assess the potentiality of transovarial transmission of these rickettsial organisms. Methods Ticks were collected from cattle in a farm in Yueyang City and the tick DNA was used as template to amplify the htrA, rrs, gltA, ompA and ompB genes of Rickettsia as well as rrs and groEL genes of Anaplasma and Ehrlichia. Results All ticks (465) collected were the cattle tick, Rhipicephalus microplus. PCR showed the minimum infection rate (MIR) was 1.5% (7/465) for Candidatus Rickettsia xinyangensis, 1.9% (9/465) for C. Anaplasma boleense, 1.3% (6/465) for Anaplasma platys, 0.6% (3/465) for A. marginale, and 1.17% (2/465) for each of A. bovis, Ehrlichia minasensis, and a non-classified Ehrlichia sp. A human pathogen, C. Rickettsia xinyangensis and A. platys were detected in 100% (3/3) and 33.3% (2/6) laboratory-hatched larval pools from infected females respectively. Conclusion Our study revealed a diversity of pathogenic rickettsial species in R. microplus ticks from Hunan Province suggesting a threat to people and animals in China. This study also provided the first molecular evidence for the potential transovarial transmission of C. Rickettsia xinyangensis and A. platys in R. microplus, indicating that R. microplus may act as the host of these two pathogens.


Introduction
Ticks are obligate blood-sucking ectoparasites that parasitize on animals and occasionally humans [1].Ixodidae ticks have been documented to transmit more than 170 pathogens worldwide, including viruses, rickettsiae, spirochetes, and protozoa [2].These pathogens can result in severe human diseases such as tick-borne forest encephalitis, severe fever with thrombocytopenia syndrome, spotted fever, Lyme disease, and Q fever [2][3][4][5][6].The cattle tick Rhipicephalus microplus in the family Ixodidae is widely distributed worldwide, especially in tropical and subtropical regions [7].It is regarded as the most economically important ectoparasite of cattle considering the massive economic losses it caused to the dairy and meat industries [8,9].Hunan Province in the Southern China has humid and warm weather with little seasonal variation [10].It is one of the most abundant regions of ticks with R. microplus as the dominant species [2,10].The paradox between the limited researches of tick-borne pathogens and the richness of ticks within Hunan suggests further investigation is needed.
The Rickettsiales contains a group of vector-borne gram-negative obligate intracellular bacteria [11].This order comprises two documented families (Rickettsiaceae and Anaplasmataceae) and one recently established family (Candidatus Midichloriaceae) [12].As well-known zoonotic pathogens, some species in the Rickettsiales can cause severe human diseases and extensive economic losses in animal husbandry [13,14].In the past 30 years, many novel species of the Rickettsiales have been discovered and the geographic distributions of the Rickettsiales have been expanded dramatically [13,15,16].Some species of the Rickettsiales were originally considered to be nonpathogenic, but have been found to cause diseases in humans in recent years [13,[17][18][19][20]. Obviously, the Rickettsiales pose a considerable challenge for public health and require continuous monitoring.Among these tick-borne Rickettsiales, Rickettsia spp.[21,22], Anaplasma spp.[23][24][25][26], and Ehrlichia spp.[23,26] are of great concern in Asia and Eurasia.
The pathogen-host relationship shaped by coevolution of the Rickettsiales with arthropods including massive pathogen replication, maintenance of persistent infection, as well as transstadial and transovarial transmission [27].It has been well documented that some Ixodidae ticks are able to transmit Rickettsia spp., Anaplasma spp.and Ehrlichia spp.transstadially [18,28,29].Additionally, it is widely accepted that the transovarial transmission plays an important role in the maintenance of Rickettsia spp.and Babesia spp.[30][31][32][33].Previous studies on transovarial transmission of Anaplasma spp.are scarce and controversial on the contrary.Some studies reported A. marginale and A. centrale can't be transmitted transovarially in R. microplus and R. simus respectively [34][35][36], while other studies provided the evidence for transovarial transmission of A. phagocytophilum in Dermacentor albipictus, A. marginale in R. microplus, and A. platys in R. sanguineus [37][38][39].
Therefore, we investigated the prevalence of rickettsial species in R. microplus collected from cattle in Hunan Province and determined whether the rickettsial organisms existed in tick eggs and laboratory hatched larvae.

Ethical statement
The collection of ticks for molecular detection was approved by the Ethics Committee of the Medical School, Wuhan University (WHU2020-YF0023), and any possible efforts were made to minimize the pain of animals.

Tick collection
Ticks were collected from a farm with 50 free-range cattle in Yueyang City, a prefecture city in Hunan Province in Southern China from August 5 to August 22, 2022 (Fig 1).There was a large free-range hilly area (approximately 2 km in radius) (Fig 1), which facilitated the survival of ticks and tick bites on cattle.To ensure the viability of the ticks, each tick was carefully held the head with a fine-tipped tweezer and was dragged out in the direction parallel to the host PLOS NEGLECTED TROPICAL DISEASES skin [40].The collected ticks were placed in 5 ml centrifuge tubes with a triangular-shaped ventilation hole on the cover.Each pool was labeled and recorded with corresponding information.After sampling, all ticks were kept in an incubator with 90% relative humidity at room temperature and immediately transported back to our laboratory [41].
Upon arriving at the laboratory, the fully engorged female ticks were placed in a tube individually and they oviposited in a week (Fig 2).The eggs (n = 100) of each tick were used for DNA extraction and the remaining eggs were incubated to hatch.For DNA extraction, the non-engorged or partially engorged ticks were pooled according to the sampling date, sex (adults only), and life stage [42]; the fully engorged female ticks were used individually after oviposition; the eggs (n = 100) or larvae (n = 100) from each tick were used in one pool.All ticks were stored at -80˚C before DNA extraction.

DNA extraction
Prior to DNA extraction, samples were rinsed with 75% ethanol for 5min, then washed and soaked with pure water to remove surface impurities and residual ethanol.The samples in the 2-ml Eppendorf tubes were immersed in liquid nitrogen to make the specimen brittle.One magnetic bead with 200 μl deionized water, 200 μl Buffer gA1 and 20 μl Proteinase K was added to each tube.Samples were ground with a frozen mixed grinding apparatus (Retsch, Germany) for 10min and centrifuged at 12,000 g for 5min.DNA was extracted from supernatant with a Trelief Animal Genomic DNA Kit (Tsingke Biotechnology, Beijing, China) and stored at -80˚C.

Tick samples
In total, 465 ticks were collected, including 379 adults, 72 nymphs, and 14 larvae.1).They had a dentition in the typical 4/4 column arrangement and a palpal article 1 ventrally lacking a ventrointernal protuberance bearing setae [42,51,52].Male R. microplus ticks carried a typical caudal appendage on the ventral plates [42] (Fig 3).The partial 16S rRNA gene sequences obtained from these samples shared 100% identities with those of R. microplus in the GenBank.Phylogenetic analysis indicated that R. microplus of this study were clustered with those from China and India in the R. microplus clade B sensu [53], which was sister to R. annulatus (Fig 5).R. microplus from Africa, Americas and Southeast Asia formed

Detection of Rickettsiales species in ticks
One Rickettsia species was identified with PCR in the ticks with a minimum infection rate (MIR) at 1.5% (7/465) (Table 2).The MIR was calculated by assuming only one tick was infected in a positive pool [45].Rickettsia positive samples comprised 3 engorged adult females, 1 larval pool, 1 nymphal pool, and 2 non-fully-engorged adult female pools (S2 Dataset).DNA sequencing showed that the Rickettsia from the ticks was most closely related to C. Rickettsia xinyangensis, which was firstly detected from patients in central China [54].The highest identities between DNA sequences from the R. microplus ticks and those of C. Rickettsia xinyangensis were all 100% on htrA, rrs, ompA, gltA and ompB genes (Table 3).Among these known identical sequences of C. Rickettsia xinyangensis, ompA gene sequence (KU853021) and gltA gene sequence (KU853023) were amplified from patients, while htrA gene sequence (KY617773), rrs gene sequence (KY617772), and ompB gene sequence (KY617776) were obtained from Haemaphysalis longicornis ticks around the patients' residences.
Four Anaplasma species were amplified from the ticks with PCR.Six engorged female adults were PCR positive for A. platys.The MIR for A. platys in the ticks was 1.3% (6/465) (Table 2).The sequence homology between the A. platys in this study and those in the Gen-Bank was 100% for rrs and 84.22%-100% for groEL.The sequences of A. marginale were obtained from 1 engorged female adult and 2 non-fully-engorged adult female pools (S2 Dataset).The MIR for A. marginale was 0.6% (3/465) in the ticks and the sequences from the ticks were 100% identical on both rrs and groEL genes with A. marginale sequences in the database.One engorged female adult and one tick pools containing 6 non-fully-engorged adult females were positive for A. bovis.The MIR for A. bovis was 0.4% (2/465, 1.17%) in the ticks.The sequences from the ticks shared 100% homology to those of A. bovis in the GenBank on rrs gene.C. Anaplasma boleense positive samples comprised 5 engorged female ticks and 4 nonfully-engorged female adult pools (S2 Dataset).The MIR for C. Anaplasma boleense sequences was 1.9% (9/465) in the ticks and the sequences of rrs and groEL genes from the ticks shared 99.30%-99.79%and 96.80%-100% homology, respectively with sequences of C. Anaplasma boleense in the GenBank.
Among 3 C. Rickettsia xinyangensis positive female ticks, all their egg clutches and laboratory-hatched larvae were also positive for C. Rickettsia xinyangensis; among 6 A. platys positive female ticks, 3 ticks' egg clutches and 2 ticks' larvae were also positive for A. platys.The DNA of A. marginale, A. bovis, C. Anaplasma boleense, E. minasensis, and a non-classified Ehrlichia sp. was not found in tick eggs or laboratory-hatched larvae.
The representative DNA sequences of rickettsial organisms obtained in this study have been submitted to the GenBank with accession numbers: OQ506629-OQ506639, OQ509025-OQ509032, and OR062291-OR062294.

Phylogenetic analysis
Phylogenetic analysis based on the concatenated sequences of htrA, rrs, ompA, gltA and ompB genes showed that the Rickettsia strain from 4 R. microplus ticks was identical with C.  Phylogenetic analysis indicated that the Anaplasma sequences amplified from ticks were divided into 4 different clusters in the phylogenetic trees (Fig 7).Phylogenetic trees of both rrs and groEL sequences showed that 3 clones (Y21, Y22 and 118) were clustered together with A. marginale; 3 clones (14, 82, and 121) were clustered together with A. platys; 4 clones (Y25, Y35, 126 and 129) were clustered in the same clade with C. Anaplasma boleense (Fig 7).One clone (Y39) was clustered in the same clade with A. bovis with rrs gene sequence, but we were unable to get the PCR product of the groEL gene from the ticks.
Phylogenetic analysis with the rrs and groEL gene sequences showed that the Ehrlichia sequences from the ticks were divided into two different clusters (Fig 8).One group (Y9 and

Discussion
In this study, we detected the DNA of 1 spotted fever group (SFG) rickettsiae, 4 Anaplasma species and 2 Ehrlichia species in the cattle tick R. microplus collected from cattle in a farm in Hunan Province of the Southern China, revealing a high diversity of rickettsial organisms in R. microplus.To our knowledge, only two of these seven species have been previously reported in Hunan Province [10], so this study contributes to a better understanding of the distribution of these rickettsial organisms.
Among seven rickettsial organisms, the DNA of C. Rickettsia xinyangensis and A. platys has been previously detected in patients [54,56].Additionally, A. marginale and A. bovis are known to be agent of bovine anaplasmosis, while A. platys and E. minasensis are the causative agents of canine infectious cyclic thrombocytopenia (ICCT/ CICT) and bovine ehrlichiosis, respectively.They may affect animal health and livestock husbandry though this study didn't report the infection status of animal hosts.The tick pools positive for rickettsial organisms were collected from a same herd on different dates.One possible explanation is that the cattle were infected with rickettsial organisms and the ticks got infected when they parasitized on cattle and sucked the blood; another explanation is that there were natural foci of these rickettsial organisms in the field.The application of cattle vaccines and regular cleaning of parasitic ticks are of great necessity, especially for free-range livestock keepers.Candidatus Rickettsia xinyangensis is a novel uncultured Rickettsia species identified in patients in the Central China [54].The patients had a mild fever, leukopenia, elevated hepatic enzyme levels and eschars on the body.Haemaphysalis longicornis ticks collected around the 3 patients' residences were also positive for C. Rickettsia xinyangensis suggesting their possible involvement in transmission [54].We detected C. Rickettsia xinyangensis in the tick eggs and laboratory-hatched larvae of R. microplus, indicating that C. Rickettsia xinyangensis can be transovarially transmitted in R. microplus tick.Transovarial transmission of C. Rickettsia xinyangensis has not yet reported in other tick species.Other Rickettsia species such as Rickettsia africae in Amblyomma variegatum [32], Rickettsia parkeri in Amblyomma maculatum and R. microplus [57,58], Rickettsia rickettsii in Amblyomma aureolatum [28], Rickettsia bellii in Ixodes loricatus [29], and Rickettsia parkeri strain Atlantic rainforest in Amblyomma ovale [30], have been demonstrated to be transovarially transmitted.The transovarial transmission (TOT) rate, proportion of infected females giving rise to at least one positive egg or larva [59], could be up to 100% in controlled laboratory settings [28,32,59].
Anaplasma platys was thought to be a canine pathogen causing a chronic infection with weight loss, anorexia, fever, lethargy, and lymphadenomegaly [60,61].It has also been reported to cause human infection with nonspecific clinical symptoms, including headache and muscle pains [56].Anaplasma platys sequences detected in R. microplus in this study were clustered with those from cattle (MN202021), buffalo (MN688298), and mosquitoes (KU585923), showing its wide host tropism.Unlike Rickettsia spp., transovarial transmission of most Anaplasma spp.and Ehrlichia spp. was thought to occur at low frequencies or not at all [36,62].A previous study have showed that transovarial transmission of A. phagocytophilum in Dermacentor albipictus was at the efficiency of 10%-40% [38].In this study, we showed the transovarial transmission of A. platys in the R. microplus at the rate of 33.3% from the infected females to laboratory-hatched larvae, suggesting that R. microplus may act as the host of A. platys.A previous study reported vertical transmission of A. platys in R. sanguineus under controlled laboratory conditions [37].
Ehrlichia minasensis is a novel pathogen causing fever, lethargy, thrombocytopenia and depression in bovines [63].It was first identified in cattle from Canada [64] and later in Brazil [65] and Colombia [66].Besides the American continent, it has also been detected in France [65], Ethiopia [67], South Africa [68], Pakistan [69] and Israel [70].In China, there were only two reports of E. minasensis, one detected E. minasensis in Haemaphysalis hystricis ticks from Hainan Island [71] and the other recently found this pathogen in R. microplus from Guizhou Province [72].Our study first reported the E. minasensis infection in R. microplus in Hunan Province, China.Since the evidence of transstadial transmission of E. minasensis in R. microplus was reported [73], the role of R. microplus in the transmission of this pathogen should be highly valued [74].
One limitation of our study is the small sample size of ticks and another is that we tested tick eggs and laboratory hatched larvae in pools rather than individually.Both limitations may cause bias on the prevalence and TOT rate of Rickettsia and Anaplasma in the ticks.

Conclusion
Our study revealed a diversity of pathogenic rickettsial species in R. microplus ticks from Hunan Province suggesting a threat to people and animals in China.This study also provided the first molecular evidence for the potential transovarial transmission of C. Rickettsia xinyangensis and A. platys in R. microplus, indicating that R. microplus may act as the host of these two pathogens.

Fig 1 .
Fig 1. Map of sampling site.Ticks were collected from cattle in a farm in Xi Tang Town, Yueyang City, Hunan Province in Southern China from August 5 to August 22, 2022.The map was constructed using ArcGIS 10.6 software.The basemap shapefiles were downloaded from national platform for common geospatial information services (tianditu.gov.cn).https://doi.org/10.1371/journal.pntd.0011546.g001

Fig 2 .
Fig 2. The oviposition of a female Rhipicephalus microplus.(A) Photographs taken on the 3rd and 7th day of oviposition, respectively.(B) Micrographs of tick eggs (top) and laboratory hatched larvae (bottom).
The adult ticks contained male ticks (n = 20), fully engorged female ticks (n = 135), and non-fullyengorged females (n = 224) (Fig 3).All samples were divided into two parts: fully engorged adult females (n = 135) were used separately and the rest (n = 330) were divided into 37 pools (Fig 4).Of 135 engorged adult female ticks, 105 (78%) of them laid eggs in the laboratory and the mass of egg clutches ranged from 61 to 374 mg.All 105 egg clutches hatched to larvae (Fig 2).All ticks were preliminary morphologically identified as R. microplus and then molecularly confirmed by amplification and sequencing of endogenous tick 16S rRNA gene (Fig 3 and Table

Fig 5 .
Fig 5. Phylogenetic tree of Rhipicephalus microplus ticks.The tree was constructed using the partial 16S rRNA gene (229 bp) of ticks based on the Maximum Likelihood (ML) method with the Kimura 2-parameter model in MEGA 7.0.Rhipicephalus sanguineus sequence was used for outgroup in the tree.The R. microplus sequences obtained in this study were marked with dots and have been submitted to the GenBank with accession numbers: OQ975295-OQ975297.https://doi.org/10.1371/journal.pntd.0011546.g005 Rickettsia xinyangensis and formed a monoclade with C. Rickettsia xinyangensis and C. Rickettsia longicornii (Fig 6).

Fig 7 .Fig 8 .
Fig 7. Phylogenetic trees of Anaplasma based on the partial rrs (470 bp) gene and groEL (205 bp) genes.Phylogenetic trees were constructed based on the Maximum Likelihood (ML) method with the Kimura 2-parameter model in MEGA 7.0.Bootstrap values (inferred from 1,000 replicates) more than 60% were indicated.Wolbachia pipientis sequences were used for outgroup in the trees.The Anaplasma sequences obtained in this study were marked with dots.https://doi.org/10.1371/journal.pntd.0011546.g007

Table 1 . PCR primers used to amplify Rickettsia, Anaplasma and Ehrlichia species.
[44]products were electrophoresed on 1.0% gel and DNA bands with expected size were excised from gels and purified with a gel extraction kit (Tsingke Biotechnology, Beijing, China).The purified DNA was cloned into the pMD19-T vector (TaKaRa, Beijing, China) and transformed into Escherichia coli DH5α competent cells.Positive clones were sequenced (Sangon, Shanghai, China).The chromatograms of DNA sequences were analyzed with Chromas (Technelysium, Tewantin, QLD, Australia) for accuracy.TheNucleotide Basic Local Alignment Search Tool (BLASTn) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was applied to compare the sequences from this study with those in the GenBank for species identification.Sequences of interest were imported into Molecular Evolutionary Genetics Analysis (MEGA) software (version 7.0) for alignment and editing.Phylogenetic trees were constructed based on the Maximum Likelihood (ML) method with the Kimura 2-parameter model, and bootstrap values were inferred from 1,000 replicates[44].

Table 3 . The rickettsial sequences in the GenBank sharing the highest homology with the rickettsial sequences obtained in this study. Species Known gene sequences with highest identity (Identity rate)
Note: C. = Candidatus.https://doi.org/10.1371/journal.pntd.0011546.t003