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We collected 1,671 Dermacentor reticulatus ticks from 17 locations in the Czech Republic, Slovakia, and Hungary. We found 47.9% overall prevalence of Rickettsia species in ticks over all locations. Sequence analysis confirmed that all tested samples belonged to R. raoultii, the causative agent of tick-borne lymphadenopathy.

Background Over 60 years ago clinical patterns resembling tick-borne rickettsioses have been described for the first time in Kazakhstan. Since 1995 the incidence of clinical cases of tick-borne rickettsioses in humans seems to be rising but studies on epidemiological data regarding the occurring etiological agents, tick vector species, prevalence and distribution throughout Kazakhstan are still scarce to date. The aim of the study was molecular investigation of ticks for spotted-fever group rickettsiae in the endemic Kyzylorda region and the so far considered as non-endemic Almaty region. A total of 2341 ticks was collected in the two regions in Kazakhstan and sorted in 501 pools: Ixodes persulcatu s (243); Dermacentor marginatus (129); Haemaphysalis punctata (104); Hyalomma asiaticum (17); Dermacentor reticulatus (3); and Rhipicephalus turanicus (5). Pools were tested for Rickettsia spp. using real-time PCR. For positive samples multilocus sequence typing (MLST) was performed. Results The calculated minimum infection rate (MIR) for rickettsiae in the investigated ticks in Almaty region varied between 0.4–15.1% and 12.6–22.7% in the Kyzylorda region. At least four different Rickettsia species were identified in the two selected regions of Kazakhstan. Two of these are already known to science: Rickettsia raoultii and R. slovaca , the latter being reported for the first time in Almaty region One new form, “ Candidatus R. yenbekshikazakhensis”, was described by MLST of six gene fragments in Almaty region and one new genotype, “genotype R. talgarensis” was detected using three gene fragments. Conclusions Kazakh physicians should be aware of rickettsioses after tick bites in both regions studied. Both, R. raoultii and R. slovaca should be included in the diagnostics. The role for human diseases has further to be investigated for the newly described rickettsiae, “ Candidatus R. yenbekshikazakhensis” and “Genotype R. talgarensis”.

Background Hard ticks act as arthropod vectors in the transmission of human and animal pathogens and are widely distributed in northern China. The aim of this study is to screen the important tick-borne pathogens (TBPs) carried by hard ticks in Inner Mongolia using metagenomic next-generation sequencing (mNGS) and to estimate the risk of human infection imposed by tick bites. Methods The adult Dermacentor nuttalli ( n  = 203) and Ixodes persulcatus ( n  = 36) ticks feeding on cattle were collected. The pooled DNA samples prepared from these ticks were sequenced as the templates for mNGS to survey the presence of TBPs at the genus level. Individual tick DNA samples were detected by genus--specific or group-specific nested polymerase chain reaction (PCR) of these TBPs and combined with DNA sequencing assay to confirm the results of mNGS. Results R. raoultii (45.32%, 92/203), Candidatus R. tarasevichiae (5.42%, 11/203), Anaplasma sp. Mongolia (26.60%, 54/203), Coxiella- like endosymbiont (CLE) (53.69%, 109/203), and Babesia venatorum (7.88%, 16/203) were detected in D. nuttalli , while R. raoultii (30.56%, 11/36), Anaplasma sp. Mongolia (27.80%, 10/36), and CLE (27.80%, 10/36) were detected in I. persulcatus . The double- and triple-pathogen/endosymbiont co-infections were detected in 40.39% of D. nuttalli and 13.89% of I. persulcatus , respectively. The dual co-infection with R. raoultii and CLE (14.29%, 29/203) and triple co-infection with R. raoultii , Anaplasma sp. Mongolia, and CLE (13.79%, 28/203) were most frequent in D. nuttalli . Conclusions This study provides insight into the microbial diversity of D. nuttalli and I. persulcatus in Inner Mongolia, China, reporting for the first time that Candidatus R. tarasevichiae had been found in D. nuttalli in China, and for the first time in the world that Anaplasma sp. Mongolia has been detected in I. persulcatus . This study proves that various vertically transmitted pathogens co-inhabit D. nuttalli and I. persulcatus , and indicates that cattle in Inner Mongolia are exposed to several TBPs. Graphical Abstract

A molecular surveillance of tick-borne diseases was performed in Hulunbuir City, Inner Mongolia. A total of 149 ticks including three species ( Ixodes persulcatus , Haemaphysalis concinna , and Dermacentor silvarum ) were collected. As many as 11 tick-borne bacterial pathogens were identified in them. Some of them have high positive rates. For example, Candidatus Rickettsia tarasevichiae was detected with a high prevalence of 72.48%, while Candidatus Lariskella sp. was detected in 31.54% of ticks. For both Rickettsia raoultii and Anaplasma phagocytophilum , two distinct genotypes were identified based on their phylogenetic trees based on 16S rRNA, gltA , and groEL sequences. Remarkable genetic diversity was also observed for 16S and flaB genes of Borreliella garinii , an agent of Lyme disease. Rickettsia heilongjiangensis causing Far-Eastern spotted fever (2.68%, 4/149), Ehrlichia muris causing human ehrlichiosis (4.70%, 7/149), Borrelia miyamotoi causing relapsing fever (2.01%, 3/149), and Borreliella afzelii causing Lyme disease (2.01%, 3/149) were also detected. Additionally, a previously uncharacterized Anaplasma species closely related to Anaplasma ovis was identified. Herein we name it “ Candidatus Anaplasma mongolica”. Based on these results, we propose that Yakeshi City might be a potential hotspot of tick-borne diseases.

Ixodid ticks transmit many obligate intracellular Rickettsial species. Several previous studies have identified Rickettsia species in the northeastern and southern part of China, but few reports on the prevalence of infection of spotted fever group Rickettsiae (SFGR) in ticks in southwest China are available. Here, we investigated SFGR in 394 adult ticks of five species including Dermacentor nuttalli, Dermacentor silvarum, Haemaphysalis longicornis, Ixodes sinensis and Ixodes persulcatus, collected in the border region between China and Burma in Yunnan Province. PCR was used to detect the presence of the citrate synthase (gltA) gene of Rickettsia species. SFGR was found in 12.1% (15/124) of I. persulcatus ticks, which was significantly higher than the 7.2% (7/97) positive D. nuttalli, 5.4% (3/56) D. silvarum, 5.6% (4/72) H. longicornis and 4.4 (2/45) I. sinensis. A portion of the gltA and ompA gene data subjected to phylogenetic analysis revealed that the detected SFGR clustered into two species, Rickettsia raoultii and the new Rickettsia species Candidatus Rickettsia jingxinensis. Detection of both Rickettsia spp. in this region indicates a potential public health threat posed by SFGR infection in Yunnan Province.

Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infection through indirect detection of specific antibodies. Previous studies have shown that Rickettsia raoultii ( R . raoultii ), a member of the SFG rickettsiae, occurs with increasing incidence in remote countries. Therefore, a rapid detection method for R . raoultii is in urgently need. In this study, a R . raoultii diagnosis method by closed dumbbell-mediated isothermal amplification (R-CDA) assay targeting a conserved sequence of the outer membrane protein A ( OmpA ) gene with high sensitivity and specificity was developed. This assay offered a rapid and simple method for on-site detection of R . raoultii . Firstly, four pairs of R-CDA primers were designed and the optimum primer set was selected to amplify target gene specifically and effectively. Then, a pair of outer primer was designed to accelerate the reaction based on the inner primers to establish the RO-CDA reaction. In addition, the results of real-time amplification curves, melting curves and end-point colorimetric judgements showed that the established visual RO-CDA reaction could accurately detect R . raoultii without cross-reaction with other closely related pathogens. Furthermore, the detection limit of visual RO-CDA assay was 10 copies/μL, which was feasible for on-site detection with merits of easy-operation, rapidity, high sensitivity, and specificity. In conclusion, the developed RO-CDA detection method could be helpful for pathogen screening and epidemic prevention at the point of care.

Background Vector-/tick-borne pathogens (V/TBPs) pose a potential threat to human and animal health globally. Information regarding canine V/TBPs is scarce and no specific study has been conducted so far to explore the microbial diversity within ticks infesting dogs from Pakistan. Herein, this knowledge gap is addressed by assessing the genetic diversity and prevalence pattern of V/TBPs in ixodid ticks with special implications for public and canine health. Methods A total of 1150 hard ticks were collected from 300 dogs across central Khyber Pakhtunkhwa (KP), Pakistan. After morpho-molecular identification, 120 tick samples were screened for the presence of V/TBPs by amplifying 16S rRNA / gltA ( Rickettsia / Ehrlichia and Wolbachia sp.), 18S rRNA ( Theileria sp.) and cox1 ( Dirofilaria sp.) genes through PCR followed by sequencing and phylogenetic study. Results In toto, 50 ixodid ticks (50/120, 41.7%) were found positive for V/TBPs DNA. The detected V/TBPs were categorized into five genera and eight species, viz. Ehrlichia ( E. canis and Ehrlichia sp.), Rickettsia ( R. massiliae, R. raoultii and Rickettsia sp.), Theileria ( T. annulata ), Dirofilaria ( D. immitis ) and Wolbachia ( Wolbachia sp.). The pathogen prevalence patterns showed that R. massiliae was the most prevalent zoonotic V/TBP (19.5%), followed by E. canis (10.8%), Rickettsia sp. (7.5%) , R. raoultii (6.7%), T. annulata (5.8%), D. immitis (5.8%), Wolbachia sp. (4.2%) and Ehrlichia sp. (3.3%), respectively. Among the screened tick species, most Rhipicephalus sanguineus sensu lato samples were found positive for V/TBP DNA (20/20,100%) followed by Rh. turanicus sensu stricto (13/20, 65%), Hyalomma dromedarii (8/20, 40%), Rh. haemaphysaloides (6/20, 30%), Hy. excavatum (2/20, 10%) and Rh. microplus (1/20, 5%). Co-occurrence of V/TBP was also detected in tick specimens (single V/TBP infection: 32 ticks; double and triple: 13 and 5 tick samples). The detected pathogens shared a phylogenetic relationship with similar isolates published in NCBI GenBank from Old and New World countries. Conclusion Ixodid ticks infesting dogs harbor a diverse array of V/TBPs including zoonotic agents from Pakistan. Furthermore, the presence of D. immitis in ticks that infest dogs raises the possibility that this parasite has either attained its dead-end host (i.e. the tick) while feeding on dogs or has expanded its range of intermediate/paratenic hosts. Further research work is needed to investigate the epidemiology and confirm the vector competence of screened tick species for these pathogens from Pakistan. Graphical Abstract

Background Increasing numbers of tick-borne pathogens are being discovered, including those that infect humans. However, reports on co-infections caused by two or more tick-borne pathogens are scarce. Case presentation A 38-year-old male farmer was bitten by a hard tick, presented with fever (37.7 °C), severe headache and ejection vomiting. Lumbar puncture was performed in the lateral decubitus. The cerebrospinal fluid (CSF) was clear, and analysis showed severe increased pressure (320 mm H 2 O), mild leukocytosis (126.0 × 10 6 /L, mononuclear cells accounting for 73%) and elevated total protein concentration (0.92 g/L). Bacterial cultures of CSF and blood were negative. The diagnosis of Rickettsia raoultii and Tacheng tick virus 1 (TcTV-1) co-infection was confirmed by amplifying four rickettsial genetic markers and the partial small (S) RNA segment of TcTV-1 from the patient’s blood. The patient gradually recovered after treatment with levofloxacin and ribavirin. Conclusions This is the first reported co-infection case with fever and meningitis caused by R . raoultii and TcTV-1. It is vital to screen for multiple pathogens in tick-bitten patients, especially in those with severe complex symptoms.

Spotted fever group (SFG) rickettsiae cause infection in humans, domestic animals and wildlife. To date, no rickettsial agents have been reported in hard ticks from the long-tailed ground squirrel (Spermophilus undulatus). A total of 50 adult ticks and 48 nymphs were collected from S. undulatus in the border region of northwestern China. Tick species (identified according to morphological and molecular characteristics) included Dermacentor nuttalli, Dermacentor silvarum and Ixodes kaiseri. Based on the cytochrome c oxidase subunit I (COI) haplotype analysis, I. kaiseri from S. undulatus belongs to an ancestral. In addition, all tick samples were analyzed for the presence of rickettsiae by PCR amplification and sequencing of six genetic markers. Rickettsia raoultii and Rickettsia sibirica subsp. sibirica were shown to occur in adults and nymphs of D. nuttalli and D. silvarum. Rickettsia sibirica subsp. sibirica was also detected in an I. kaiseri adult. Dermacentor silvarum and I. kaiseri were found for the first time on S. undulatus. Rickettsia raoultii and R. sibirica subsp. sibirica were detected in two Dermacentor and one Ixodes species, respectively, suggesting that these rickettsiae circulate in the region of the China-Kazakhstan border by hard ticks infesting S. undulatus.

Background Melophagus ovinus (Diptera: Hippoboscidae), a hematophagous ectoparasite, is mainly found in Europe, Northwestern Africa, and Asia. This wingless fly infests sheep, rabbits, and red foxes, and causes inflammation, wool loss and skin damage. Furthermore, this parasite has been shown to transmit diseases, and plays a role as a vector. Herein, we investigated the presence of various Rickettsia species in M. ovinus . Methods In this study, a total of 95 sheep keds were collected in Kuqa County and Alaer City southern region of Xinjiang Uygur Autonomous Region, northwestern China. First, collected sheep keds were identified on the species level using morphological keys and molecular methods based on a fragment of the 18S ribosomal DNA gene (18S rDNA). Thereafter, to assess the presence of rickettsial DNA in sheep keds, the DNA of individual samples was screened by PCR based on six Rickettsia -specific gene fragments originating from six genes: the 17-kilodalton antigen gene ( 17-kDa ), 16S rRNA gene ( rrs ), surface cell antigen 4 gene ( sca4 ), citrate synthase gene ( gltA ), and outer membrane protein A and B genes ( ompA and ompB ). The amplified products were confirmed by sequencing and BLAST analysis ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ). Results According to its morphology and results of molecular analysis, the species was identified as Melophagus ovinus , with 100% identity to M. ovinus from St. Kilda, Australia (FN666411). DNA of Rickettsia spp. were found in 12 M. ovinus samples (12.63%, 12/95). Rickettsia raoultii and R. slovaca were confirmed based on phylogenetic analysis, although the genetic markers of these two rickettsial agents amplified in this study showed molecular diversity. Conclusions This is the first report of R. raoultii and R. slovaca DNA in M. ovinus. Rickettsia slovaca was found for the first time around the Taklimakan Desert located in China. This finding extends the geographical range of spotted fever group rickettsiae .