Candidatus Neoehrlichia mikurensis is widespread in questing Ixodes ricinus ticks in the Czech Republic

Journal Pre-proof Candidatus Neoehrlichia mikurensis is widespread in questing Ixodes ricinus ticks in the Czech Republic Jaroslav Ondruˇs (Conceptualization) (Funding acquisition) (Methodology) (Resources) (Investigation) (Data curation) (Writing ´ zova´ (Resources), Vojtech original draft) (Visualization), Alena Balaˇ ´ z (Resources), Krist´ına Zechmeisterova´ (Resources) (Formal Balaˇ ˇ analysis), Adam Novobilsk´y (Formal analysis), Pavel Sirok´ y (Resources) (Writing - review and editing) (Supervision)

PII:

S1877-959X(19)30315-2

DOI:

https://doi.org/10.1016/j.ttbdis.2020.101371

Reference:

TTBDIS 101371

To appear in:

Ticks and Tick-borne Diseases

Received Date:

23 July 2019

Revised Date:

3 January 2020

Accepted Date:

8 January 2020

´ zova´ A, Balaˇ ´ z V, Zechmeisterova´ K, Novobilsk´y A, Please cite this article as: Ondruˇs J, Balaˇ ˇ Sirok´ y P, Candidatus Neoehrlichia mikurensis is widespread in questing Ixodes ricinus ticks in the Czech Republic, Ticks and Tick-borne Diseases (2020), doi: https://doi.org/10.1016/j.ttbdis.2020.101371

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Candidatus Neoehrlichia mikurensis is widespread in questing Ixodes ricinus ticks in the Czech Republic

Jaroslav Ondruša,b,*, Alena Balážováb, Vojtech Balážb, Kristína Zechmeisterováb, Adam Novobilskýc, Pavel Širokýa,b

CEITEC - Central European Institute of Technology, University of Veterinary and Pharmaceutical

Sciences Brno, Palackého tř. 1946/1, 612 42 Brno, Czech Republic b

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a

Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University

Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Hudcova 296/70,

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c

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of Veterinary and Pharmaceutical Sciences, Palackého tř. 1946/1, 612 42 Brno, Czech Republic

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621 00 Brno, Czech Republic

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*Corresponding author. Tel: +420 776 460 514 E-mail address: [email protected]

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E-mail addresses of other authors: AB, [email protected]; VB, [email protected];

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KZ, [email protected]; AN, [email protected]; PŠ, [email protected]

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Abstract Candidatus Neoehrlichia mikurensis, the causative agent of tick-borne “neoehrlichiosis” has recently been reported in humans, mammals and ticks in Europe. The aim of this study was to map the distribution of this bacterium in questing ticks in the Czech Republic. A total of 13,325 Ixodes ricinus including 445 larvae, 5270 nymphs and 7610 adults were collected from vegetation by flagging in 140 Czech towns and villages from every region of the Czech Republic. The ticks were pooled into 2665 groups of 5 individuals respecting life stage or sex and tested for the presence of Ca. Neoehrlichia

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mikurensis by conventional PCR targeting of the groEL gene. The bacterium was detected in 533/2665 pools and 125/140 areas screened, showing an overall estimated prevalence of 4.4% in

ticks of all life stages. Phylogenetic analysis revealed only small genetic diversity among the strains

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found. Two pools of questing larvae tested positive, suggesting transovarial transmission. According to this study, Ca. Neoehrlichia mikurensis is another tick-borne pathogen widespread in I. ricinus ticks

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in the Czech Republic.

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Abbreviations:

GTR, General Time Reversible; MIR, Minimum infection rate; CI, Confidence intervals; pos/ex,

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numbers of positive/examined pools of ticks; TBEV, Tick-borne encephalitis virus; s. l., sensu lato

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Keywords: Candidatus Neoehrlichia mikurensis; Tick-borne disease; Anaplasmataceae; Ixodes ricinus;

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Czech Republic

1.

Introduction

Candidatus Neoehrlichia mikurensis is an emerging tick-borne intracellular bacterium in the family Anaplasmataceae, order Rickettsiales. Ticks of the genus Ixodes are considered the main vector in Europe (Portillo et al., 2018) and wild rodents serve as reservoirs (Burri et al., 2014). Ca. Neoehrlichia 2

mikurensis has been detected in several rodent species which inhabit the Czech Republic, e. g. Apodemus flavicollis, A. agrarius, Microtus agrestis, Myodes glareolus and Erinaceus roumanicus (Anděra, 2019; Földvári et al., 2014; Krücken et al., 2013; Silaghi et al., 2012). Even though this bacterium was discovered in 1999 in Ixodes ricinus ticks collected from roe deer (Capreolus capreolus) in the Netherlands (Schouls et al., 1999), it was only recognized as a member of a new genus in 2004, when it was isolated from brown rats (Rattus norvegicus) captured on Mikura Island, Japan (Kawahara et al., 2004).

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Ca. Neoehrlichia mikurensis may cause “neoehrlichiosis” in humans. The first report of this came from Sweden, where a 77-year-old man developed severe symptoms which were proven to be

related to a Ca. Neoehrlichia mikurensis infection (Welinder-Olsson et al., 2010). Since then, other

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cases of neoehrlichiosis have been described in European countries, including the Czech Republic (Pekova et al., 2011). Manifestations of neoehrlichiosis are atypical. The course of the infection

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ranges from asymptomatic infection (Welc-Falȩciak et al., 2014b) or mild illness (Li et al., 2012) to serious life-threatening disease (von Loewenich et al., 2010; Welinder-Olsson et al., 2010). Fever,

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headache, malaise, myalgia, arthralgia and vascular events have been observed (Silaghi et al., 2016). Vascular endothelial cells were identified as the target of infection (Wass et al., 2019). The most

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severe progress of neoehrlichiosis occurs in immunocompromised and splenectomized individuals (Grankvist et al., 2014). The only animal that has been shown to develop symptomatic neoehrlichiosis

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is the dog (Diniz et al., 2011). In the case of rodents (R. norvegicus), no clinical signs of the disease

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have been observed (Kawahara et al., 2004). Ca. Neoehrlichia mikurensis has been reported in questing ticks in 18 European countries. A detailed review can be found in Portillo et al. (2018). In Central Europe, this bacterium has been detected in host-seeking ticks in every country with the exception of Slovenia. Greatly varying prevalences have been observed in countries neighboring the Czech Republic. On the one hand, low prevalences have been detected in Poland, where only about 0.2% of questing I. ricinus adults and nymphs were found

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to be infected (Welc-Falȩciak et al., 2014a). On the other hand, high prevalences have been detected in several areas of Austria and Germany. In total, 23.5% of host-seeking I. ricinus of all life stages harbored this bacterium in Kundl, Austria (Derdáková et al., 2014). Furthermore, at least 25.8% of questing I. ricinus adults and nymphs were found to be infected in Leipzig and 33.9% in Saarland in Germany (Silaghi et al., 2012). In the Czech Republic, Ca. Neoehrlichia mikurensis has already been detected in seven locations, which were tested in four studies (Derdáková et al., 2014; Richter and Matuschka, 2012; Venclikova et al., 2014; Venclíková et al., 2016). These publications, however, only

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investigated a relatively small number of ticks or were focused on comparing infection ratios among different habitats. Due to this, our goal was to map the current spread of Ca. Neoehrlichia mikurensis

Materials and methods

2.1.

Sampling

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in questing I. ricinus ticks throughout the Czech Republic.

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Questing ticks were collected from vegetation by flagging in the suburban areas and areas surrounding Czech cities, towns and villages (n = 140) throughout the country during the years 2016

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to 2018. The settlements chosen were inhabited by 15,000 or more people or were spots of high touristic activity. Majority of locations were flagged by one person for 60 minutes or by two people

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for 30 minutes using a white denim squares 1 m × 1 m attached to a wooden stick as a flag (Tkadlec et al., 2018). During sampling, only I. ricinus and adult Dermacentor reticulatus were found. However,

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D. reticulatus were excluded from our study and not further analyzed, because they had only been found in 3/140 locations. Furthermore, this species does not seem to have epidemiological relevance (Krücken et al., 2013; Richter et al., 2013) in the transmission of Ca. Neoehrlichia mikurensis. The ticks were placed into test tubes with 70% ethanol, transported to the laboratory and sorted by species, sex and life stage using the Olympus SZX16 stereomicroscope following the available keys (for example Nosek and Sixl, 1972). However, ticks were not tested for being I. inopinatus (Chitimia4

Dobler et al., 2018; Estrada-Peña et al., 2014). Samples were stored in a refrigerator at 4 oC until DNA extraction.

2.2.

DNA extraction

The ticks were pooled into groups containing 5 specimens of the same sex or life stage. No pools of mixed sex or life stages were made, and every pool contained exactly 5 individuals. DNA was

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extracted using a commercial NucleoSpin® Tissue kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and following the manufacturer's protocol. In the pre-lysis step, the pooled ticks were homogenized using 1 g of 1.4 mm Silica beads (BIOplastics, Landgraaf, the Netherlands) in the

MagNA Lyser (Roche Diagnostics Ltd., Rotkreuz, Switzerland) for 99 seconds at 7, 000 speed. The

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purity and concentration of the extracted DNA were checked by an Implen NanoPhotometer® P330

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(Implen, Munich, Germany). The typical DNA yield ranged from 12 to 18 μg, 5 to 7 μg, 2 to 4 μg and 1 to 2 μg for adult females, adult males, nymphs and larvae, respectively. Extracted DNA samples were

PCR analysis

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2.3.

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stored at -20 °C until the PCR detection.

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A new conventional PCR reaction targeting the groEL gene (heat shock protein) was used to detect the DNA of Ca. Neoehrlichia mikurensis in our samples. Primers were designed with the Geneious

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11.1.4 software (https://www.geneious.com) using relevant sequences available in the GenBank. Primers CNM_groEL_PCR_F (5'-AACTACAACATGTTCTATTTTAACAGC-3') and CNM_groEL_PCR_R (5'TCGTCATTAATAACGTATTTTGCACC-3') amplified the 654-bp-long fragment of the groEL gene. The primer annealing temperature was established by a gradient PCR. Optimizing PCR runs contained positive (DNA extracted from the I. ricinus which had tested positive for Ca. Neoehrlichia mikurensis by an unrelated PCR and further confirmed by sequencing) and negative controls (PCR water). The

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specificity of the reaction was confirmed by sequencing. The PCR reaction was carried out in 25 μl containing: 2x PPP Master Mix (Top-Bio s.r.o., Vestec, Czech Republic), 500 nM of forward and reverse primer, PCR water and 2 μl of the template DNA solution. The following PCR scheme was used: initial denaturation at 94 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 52.4 °C for 15 s and extension at 72 °C for 45 s. The final extension took 7 min at 72 °C. The PCR products were separated using electrophoresis on 1.5 % agarose gels stained with Midori

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Green (Elisabeth Pharmacon, Brno, Czech Republic) and visualized under UV light.

Sequence analysis

Positive samples from the 10 localities with the highest Ca. Neoehrlichia mikurensis prevalence and

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from the surroundings of the 10 most populated towns and cities (specified in Supplementary Table

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S1) were sequenced from both ends with the same primers that were used for the PCR detections. Specific PCR products of 654 bp were cut from the gel, extracted using a Gel/PCR DNA Fragments Kit

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(Geneaid Biotech Ltd., Taipei, Taiwan) and sent for Sanger sequencing (Macrogen, Amsterdam, the Netherlands). The sequences were trimmed and aligned manually with Geneious 11.1.4. Next,

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relevant sequences were found in the GenBank database using blast tool in BLAST+ 2.9.0 program package (Zhang et al., 2000). The sequences obtained and downloaded were aligned using MUSCLE

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3.8.425 Alignment (Edgar, 2004). The final untrimmed alignment of 1447 bp contained 35 sequences (6 from this study). A sequence from Anaplasma phagocytophilum (AF383225) was selected as an

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outgroup. Bayesian inference analysis was performed by the Geneious 11.1.4 plugin MrBayes 3.2.6. (Huelsenbeck and Ronquist, 2001) using the GTR+Γ substitution model for 1.1*106 generations with a subsampling frequency of 200 and a burn-in length 105. Maximum likelihood phylogenetic analysis was performed by the Geneious 11.1.4 plugin PhyML 3.3.20180621 (Guindon et al., 2010) using the GTR+Γ+I substitution model with parameters estimated from the input data. The GTR+Γ+I substitution model was chosen as the best by the Smart Model Selection (Lefort et al., 2017)

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software available online [http://www.atgc-montpellier.fr/phyml-sms/]. Nodal supports were calculated with 1,000 bootstrap replicates. The tree was visualized using TreeGraph 2.14.0-771 beta (Stöver and Müller, 2010) and edited in Adobe Illustrator CS6. Genetic distances of the unique sequences to the most similar sequence (EU810407) found in the GenBank were calculated in MEGA X (Kumar et al., 2018) and described as p-values.

Statistical analysis

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2.5.

Minimum infection rates (MIR) were calculated as the ratio of the number of positive pools to the

total number of ticks tested. Estimated prevalence and 95% confidence intervals (CI) were calculated

EpiTools epidemiological calculator available online

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using the “Perfect test and exact confidence limits” method based on Cowling et al. (1999) on the

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[http://epitools.ausvet.com.au/content.php?page=PPFreq1]. The distribution map of Ca. Neoehrlichia mikurensis in I. ricinus in the Czech Republic was constructed using QGIS 3. 4. 4-Madeira

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(QGIS Development Team, 2019). The Pearson's goodness of fit chi-square test was calculated in Microsoft Excel and Fisher's 2 x 2 exact test was calculated online using the Easy Fisher Exact Test

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Calculator [https://www.socscistatistics.com/tests/fisher/default2.aspx] when applicable. A

Results

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3.

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significance level of =0,05 was considered the limiting probability for both tests.

3.1.

Prevalence and distribution of Ca. Neoehrlichia mikurensis

A total of 13,325 questing I. ricinus ticks, including 445 larvae, 5270 nymphs and 7610 adults were collected. Overall a total of 2665 pools, including 89 pools of larvae, 1054 pools of nymphs, 736 pools of adult females and 786 pools of adult males were made. Altogether 533 out of the 2665 pools (20.0%) contained one or more infected ticks. The calculated prevalences and MIR regarding tick life 7

stages and sex are described in Table 1. The prevalences between all four groups of ticks, calculated on the basis of overall positive to negative pool numbers, did not differ significantly (P=0.080). The MIRs of male and female adults were similar. Adult ticks were more frequently infected than nymphs (p<0.001) and larvae (p<0.001). Nymphs were more frequently infected than larvae (p<0.001). The total MIR and calculated prevalences of Ca. Neoehrlichia mikurensis in the entire Czech Republic in ticks of all life stages (including larvae) were 4.0% and 4.4% (95% CI: 4.0–4.7%), for nymphs 3.4% and 3.6% (95% CI: 3.1-4.2%) and for adults 4.6% and 5.1% (95% CI: 4.6-5.7%), respectively (Table 1). The

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ratio of positive to negative tick pools in the individual regions differed significantly (p<0.001). The regions with the highest calculated prevalences for ticks of all life stages (including larvae) were the Jihočeský Region (7.6%, 95% CI: 6.0-9.5%) and the Karlovarský Region (7.6%, 95% CI: 5.1-10.7%). For

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detailed information regarding prevalences and MIR of adults and nymphs in individual regions, see Table 2.

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Ca. Neoehrlichia mikurensis was detected in 125/140 of the areas screened. Larvae data were excluded from the calculations of MIR and prevalences in the localities sampled, because larvae were

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sampled only in 14/140 locations. The highest prevalence in ticks of all life stages was observed in the area surrounding Český Krumlov (24.2%; 95% CI: 12.1–40.8%), where 15 nymphs and 65 adults were

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collected. For the full list of all 140 sampling sites, with detailed information regarding coordinates, districts, regions, numbers of positive and tested pools of nymphs, adults, and calculated MIR see

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Supplementary Table S1. The distribution map of Ca. Neoehrlichia mikurensis in questing I. ricinus ticks (excluding larvae) in the Czech Republic, expressed by MIR per region, shows that the highest

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prevalence of the bacterium was in the western part of the Czech Republic (Fig. 1).

3.2

Variability of groEL gene sequences

The sequencing of 20 samples and subsequent BLAST confirmed the specificity of the PCR reaction. All the groEL sequences were more than 99% identical to the Ca. Neoehrlichia mikurensis sequence 8

from Germany (EU810407). The phylogenetic relationships of our isolates based on the partial groEL sequences are depicted in Fig. 2. Sequences identical to EU810407 were merged into “Ca. Neoehrlichia mikurensis CR Common” (Supplementary Table S1). All the sequences were clustered with those from Europe (excluding Switzerland). Unique sequences were found in Rakovník, Český Krumlov, Pardubice, Olomouc and Brno and had 0.002, 0.003, 0.002, 0.002, 0.002 p-distances to EU810407, respectively. These sequences have been deposited in GenBank and can be accessed

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under accession numbers MN151363-MN151367, respectively.

Discussion

This is the first large-scale survey of Ca. Neoehrlichia mikurensis in questing I. ricinus ticks in the

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Czech Republic. We detected this bacterium on majority of screened localities (125/140). The

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estimated prevalences in the host-seeking I. ricinus of all life stages (including larvae), adults and nymphs in the Czech Republic was 4.4%, 4.6% and 3.4%, respectively.

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In the Czech Republic, four studies have reported host-seeking ticks infected with Ca. Neoehrlichia mikurensis in all seven areas screened prior to this study. However, these reports investigated only a

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small number of ticks or were not focused on spatial distribution. Firstly, Richter and Matuschka (2012) detected 2/20 (10%) infected adult I. ricinus collected from vegetation in Konopiště, Benešov

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(compared to calculated prevalence 7.8% in adults and nymphs in our study). Next, Derdáková et al. (2014) detected Ca. Neoehrlichia mikurensis in 3/138 (2.2%) I. ricinus ticks of unspecified life stages

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in Dvůr Králové (3.9% in adults and nymphs in our study). Venclikova et al. (2014) screened 1473 questing I. ricinus pooled into 320 groups, to demonstrate its prevalence in different types of ecosystems. The questing ticks were collected in natural and urban ecosystems represented by the nearby areas of Proskovice and Ostrava-Bělský Les. In these areas, their ratio of 54/320 pooled I. ricinus adults and nymphs (3-5 individuals in one pool) did not significantly differ (P=0.775) from the ratio we detected (4/22 of adults and nymphs were positive, giving the calculated prevalence 3.9%). 9

In addition, Venclíková et al. (2016) screened 2473 host-seeking I. ricinus ticks individually for the presence of Ca. Neoehrlichia mikurensis in three areas representing three different ecosystems each year during the years 2011-2014. Specimens were gathered in Valtice, Pohansko and Suchov corresponding to urban park, natural woodland and pasture ecosystems, respectively. Overall, the prevalence was 5.1% in adults and nymphs but ranged from 0.8% to 11.6% depending on the sampling year, life stage and locality. When comparing this with our findings, the calculated prevalence in the corresponding districts was 7.8% in adults and nymphs.

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In Europe, Ca. Neoehrlichia mikurensis has been reported in questing ticks from 18 countries so far with its prevalences varying from 0.1% to 24.3% (reviewed by Portillo et al., 2018). All the countries neighboring the Czech Republic are also known for the presence of Ca. Neoehrlichia mikurensis in

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ticks. To date, infected questing I. ricinus ticks of all life stages have been reported in Austria

(prevalences ranging from 4.2% to 22.1%; Derdáková et al., 2014; Glatz et al., 2014; Schötta et al.,

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2017), 2.2% - 24.2% adults and nymphs were infected in Germany (Richter and Matuschka, 2012; Silaghi et al., 2012; Obiegala et al., 2014) and 0.2% adults and nymphs in Poland (Welc-Falȩciak et al.,

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2014a). In Slovakia, 1.1% to 11.6% adults and nymphs were infected (Špitalská et al., 2008; Pangrácová et al., 2013; Derdáková et al., 2014; Blaňarová et al., 2016; Svitálková Hamšíková et al.,

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2016). Interestingly, several positive I. ricinus feeding larvae were detected on uninfected rodents, however, authors of the study assumed that the larvae acquired infection by prefeeding on an

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infected host (Svitálková Hamšíková et al., 2016). Overall, the prevalences of Ca. Neoehrlichia

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mikurensis vary greatly across Europe. Ca. Neoehrlichia mikurensis is less prevalent in ticks in Europe than Borrelia burgdorferi sensu lato (s.l.). According to the metaanalysis synthesized in 2005, the overall prevalences of Borrelia spirochetes in Europe were 18.6% and 10.1% for adults and nymphs, respectively (Rauter and Hartung, 2005). Similar prevalences have recently been reported in the ticks in the Czech Republic, where 17.3% host-seeking adults and nymphs ticks were infected with B. burgdorferi s.l. (Kybicová et

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al., 2017). Although the infection rates differ, several similarities can be noticed. The adults in our study were significantly (p<0.001) more frequently infected than the nymphs, which was also observed in the case of B. burgdorferi s.l. (Rauter and Hartung, 2005). Surprisingly, two recent studies from the Czech Republic showed the opposite trend (Hönig et al., 2015; Kybicová et al., 2017). However, it is possible that the sampled adults fed in their nymphal stage on a host incompetent for Borrelia transmission such as roe deer (Jaenson and Tälleklint, 1992). To date, no similar phenomenon has been documented for Ca. Neoehrlichia mikurensis. Next, we showed that the

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Jihočeský and Karlovarský Regions are the regions with the highest prevalence of Ca. Neoehrlichia mikurensis. Interestingly, the Jihočeský Region is already known as a high-risk area for Lyme

borreliosis. The prevalence of B. burgdorferi s.l. in adult ticks is higher in this region than in the rest

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of the country (Daniel et al., 2016). In addition, the annual incidence of Lyme borreliosis in humans in the Jihočeský Region (44.8 cases/100,000 inhabitants) is higher than the average in the Czech

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republic (37.3/100,000) (Kříž et al., 2018).

Interestingly, we have detected Ca. Neoehrlichia mikurensis in two pools of larvae. The presence of

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the bacterium in host-seeking larvae might indicate transovarial transmission. However, the possibility of premature detachment from an infected mammal during the blood meal should also be

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considered. Previously, Ca. Neoehrlichia mikurensis was detected in 4/10 questing larvae collected in Austria (Derdáková et al., 2014). Nonetheless, transovarial transmission has not yet been proven

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under controlled laboratory conditions and additional research is needed to understand the mechanism of Ca. Neoehrlichia mikurensis transmission in ticks. In rodents it is likely that

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transplacental transmission does occur, although further confirmation is needed (Obiegala et al., 2014).

According to our results, it is most likely that neoehrlichiosis is currently being misdiagnosed or not diagnosed at all and, therefore, cases of it have been missed in the Czech Republic. Even though infected ticks are common in the Czech Republic, only two cases of neoehrlichiosis have been

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reported so far (Pekova et al., 2011). This is probably the result of insufficient knowledge about the pathogen and the unavailability of diagnostic tests. As a result, human patients are not tested for this pathogen even after a tick bite. Therefore, physicians and specialists in the Czech Republic should keep neoehrlichiosis in mind, especially in cases, of immunocompromised individuals with sudden health complications. Such patients should be tested routinely for a Ca. Neoehrlichia mikurensis infection, since they constitute the group most at risk (Grankvist et al., 2014). Still, Ca. Neoehrlichia mikurensis is not likely to be an opportunistic pathogen, since cases of otherwise healthy individuals

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have also been reported (Li et al., 2012; Schwameis et al., 2016). It should be highlighted that the diagnosis and treatment of the Ca. Neoehrlichia mikurensis infection is relatively simple, as whole

blood can be used for detection by molecular techniques (Pekova et al., 2011; Quarsten et al., 2017)

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and the administration of doxycycline leads to complete recovery (Grankvist et al., 2014).

The limitations of this study lie in the sample pooling. It is not possible to detect co-infections of Ca.

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Neoehrlichia mikurensis with other tick-borne pathogens. In Europe for example, Borrelia afzelii was reported to co-infect ticks with Ca. Neoehrlichia mikurensis (Richter and Matuschka, 2012).

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Interestingly, it was later shown that the ratio of co-infections of these two pathogens is above the expected level that would result from a random co-occurrence (considering the prevalence of both

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pathogens mentioned (Andersson et al., 2013)), suggesting mutual reservoir hosts. This drawback caused by the pooling is, however, compensated for by the number of ticks collected. Our results

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should be treated carefully, since our study does not provide accurate data about the prevalence of Ca. Neoehrlichia mikurensis, but rather shows the large-scale distribution of this agent in questing

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ticks in the Czech Republic.

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Conclusions

This study shows for the first time that the emerging bacterium Candidatus Neoehrlichia mikurensis is widespread among ticks in the Czech Republic with the overall prevalence of 4.4% and the greatest 12

prevalence in the western part of the country. Due to the limited knowledge of epidemiological aspects and the lack of routine diagnosis of neoehrlichiosis in humans, the medical importance of Ca. Neoehrlichia mikurensis should be reconsidered in the Czech Republic.

Acknowledgements This study was funded by IGA VFU project 205/2018/FVHE formally submitted by Dana Rymešová.

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Sampled material was obtained during work on the project AZV No. 16-33934A from the Grant Agency for Health Research of the Czech Republic. Adam Novobilský was supported by the Ministry of Education, Youth and Sports, the project "FIT" (Pharmacology, Immunotherapy, nanoToxicology) number CZ.02.1.01/0.0/0.0/15_003/0000495 and project RO0519 of the Czech Ministry of

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Agriculture. Funding sources were not involved in the design of the study; in the collection, analysis

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and interpretation of the data; in the writing of the report; and in the decision to submit the article for publication. We are grateful to Charles du Parc for the English proofreading and to anonymous

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reviewers for suggestions that further improved our manuscript. Jaroslav Ondruš would also like to thank Pavel Kulich, Petr Lány, Adam Valček, Kristína Nešporová, Josef Illek, Michaela Honsová, Kamil

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Author statement

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Zeman and Branka Bilbija for their consultations and support.

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Jaroslav Ondruš: Conceptualization, Funding acquisition, Methodology, Resources, Investigation, Data Curation, Writing - Original Draft, Visualization. Alena Balážová: Resources. Vojtech Baláž: Resources. Kristína Zechmeisterová: Resources, Formal analysis. Adam Novobilský: Formal analysis. Pavel Široký: Resources. Writing - Review & Editing, Supervision.

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Declarations of interest: none

Competing interests statement

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Figures

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Fig. 1. Distribution of Ca. Neoehrlichia mikurensis in questing Ixodes ricinus in the Czech Republic.

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Fig. 2. Bayesian phylogenetic tree of Ca. Neoehrlichia mikurensis based on the untrimmed MUSCLE alignment of the groEL gene sequences. Sequences from this study are in bold. Branch lengths indicate expected numbers of substitutions per nucleotide site. Numbers show posterior probabilities under the Bayesian inference/bootstrap values for Maximum likelihood. Asterisk

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represents values below 65%.

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Table 1 Calculated prevalences and MIR of Ca. Neoehrlichia mikurensis in Ixodes ricinus ticks in relation to life

Sex/life stage

Numbers of positive to examined pools

MIR

Calculated prevalences (95% CI)

Adult females

173/736

4.7%

5.2% (4.5-6.0)

Adult males

180/786

4.6%

5.1% (4.4-5.8)

Adults

353/1522

4.6%

5.1% (4.6-5.7)

Nymphs

178/1054

3.4%

3.6% (3.1-4.2)

Larvae

2/89

0.4%

Total

533/2665

4.0%

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stage and sex.

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MIR: minimum infection rate.

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na

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95% CI: 95% Confidence interval.

24

0.5% (0.1-1.6)

4.4% (4.0-4.7)

oo

f

Table 2

Calculated prevalences and MIR of Ca. Neoehrlichia mikurensis in examined Ixodes ricinus adults and nymphs in individual regions of the Czech Republic.

MIR

Region

Adults

Nymphs

Adults + Nymphs

Adults

Jihočeský

49/142

23/77

72/219

6.9%

6.0%

Jihomoravský

25/150

16/137

41/287

3.3%

2.3%

Karlovarský

25/55

4/26

29/81

Královohradecký

9/69

7/48

16/117

Liberecký

3/39

4/43

7/82

Moravskoslezský

40/160

10/52

Olomoucký

17/130

Pardubický Plzeňský

pr

Numbers of positive to examined pools

Adults + Nymphs

Adults

Nymphs

Adults + Nymphs

6.6%

6.9% (4.4-10.1)

7.7% (6.0-9.6)

2.9%

3.2% (2.3-5.2)

2.5% (1.4-4.0)

3.0% (2.2-4.1)

8.1% (6.0-10.6)

9.1%

3.1%

7.2%

11.4% (7.4-16.5)

3.3% (0.9-8.2)

8.5% (5.7-12.0)

2.6%

2.9%

2.7%

2.7% (1.3-5.2)

3.1% (1.2-6.3)

2.9% (1.7-4.7)

1.5%

1.9%

1.7%

1.6% (0.3-4.6)

1.9% (0.5-4.9)

1.8% (0.7-3.6)

50/212

5.0%

3.8%

4.7%

5.6% (4.0-7.5)

4.2% (2.0-7.6)

5.2% (3.9-6.9)

9/58

26/188

2.6%

3.1%

2.8%

2.8% (1.6-4.4)

3.3% (1.5-6.2)

2.9% (1.9-4.2)

21/116

3/28

24/144

3.6%

2.1%

3.3%

3.9% (2.5-5.9)

2.2% (0.5-6.4)

3.6% (2.3-5.3)

21/67

13/37

34/104

6.3%

7.0%

6.5%

7.2% (4.5-10.9)

8.3% (4.4-13.8)

7.6% (5.3-10.5)

20/74

8/66

28/140

5.4%

2.4%

4.0%

6.1% (3.7-9.3)

2.6% (1.1-5.0)

4.4% (2.9-6.3)

Středočeský

54/166

28/151

82/317

6.5%

3.7%

5.2%

7.6% (5.7-9.8)

4.0% (2.7-5.8)

5.8% (4.6-7.2)

Ústecký

26/127

27/151

53/278

4.1%

3.6%

3.8%

4.5% (2.9-6.5)

3.9% (2.6-5.6)

4.1% (3.1-5.4)

24/114

8/64

32/178

4.2%

2.5%

3.6%

4.6% (3.0-6.8)

2.6% (1.1-5.1)

3.9% (2.7-5.4)

19/113

18/116

37/229

3.4%

3.1%

3.2%

3.6% (2.2-5.6)

3.3% (2.0-5.2)

3.5% (2.4-4.7)

Vysočina Zlínský

na l

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Praha

Pr

e-

Nymphs

Calculated prevalences (95% CI)

25

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MIR: minimum infection rate.

Jo ur

na l

Pr

e-

pr

95% CI: 95% Confidence interval.

26