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‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the Czech Republic
Accepted Manuscript Title: ‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the Czech Republic Author: Eva Hajduskova Ivan Literak Ivo Papousek Francisco B. Costa Marketa Novakova Marcelo B. Labruna Lenka Zdrazilova-Dubska PII: DOI: Reference:
S1877-959X(16)30018-8 http://dx.doi.org/doi:10.1016/j.ttbdis.2016.02.004 TTBDIS 610
To appear in: Received date: Revised date: Accepted date:
18-7-2014 10-1-2016 3-2-2016
Please cite this article as: Hajduskova, E., Literak, I., Papousek, I., Costa, F.B., Novakova, M., Labruna, M.B., Zdrazilova-Dubska, L.,‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the Czech Republic, Ticks and Tick-borne Diseases (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the
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Czech Republic
3 Eva Hajduskovaa,b, Ivan Literaka,c, Ivo Papouseka, Francisco B. Costad, Marketa Novakovaa,c, Marcelo B.
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Labrunad, Lenka Zdrazilova-Dubska a,e,f (ca)
ip t
4
cr
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a
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of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1/3, 612 42 Brno, Czech Republic
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b
us
Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University
Institute of Parasitology, Biology Centre, ASCR v.v.i., Branisovska 31, 370 05 Ceske Budejovice,
Czech Republic
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c
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Brno, Czech Republic
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d
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and Animal Husbandry, University of São Paulo, Avenue Orlando Marques de Paiva, 87 São Paulo,
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Brazil 05508-270
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e
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Brno, Czech Republic
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f
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Brno, Czech Republic
M
CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1/3, 614 42
te
d
Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine
Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53
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an
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Department of Pharmacology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00
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corresponding author: Lenka Zdrazilova-Dubska, Department of Laboratory Medicine, Masaryk
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Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic, +420 543 136 704,
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[email protected].
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24 Abstract:
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A novel rickettsial sequence in the citrate synthase gltA gene indicating a novel Rickettsia species has
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been detected in 7 out of 4524 Ixodes ricinus ticks examined within several surveys performed in the
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Czech Republic from 2005 to 2009. This new Candidatus Rickettsia sp. sequence has been found in 2
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nymphs feeding on wild birds (Luscinia megarhynchos and Erithacus rubecula), in a male tick from
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vegetation, and 4 ticks feeding on a dog (3 males, 1 female tick). Portions of the ompA, ompB, sca4,
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and htrA genes were not amplifiable in these samples. A maximum likelihood tree of rickettsiae
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based on comparisons of partial amino acid sequences of citrate synthase and nucleotide sequences
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of 16S rDNA genes and phylogenetic analysis revealed a basal position of the novel species in the
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proximity of R. bellii and R. canadensis. The novel species has been named ‘Candidatus Rickettsia
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mendelii’ after the founder of genetics, Gregor Mendel.
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Highlights: •
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ticks.
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A novel rickettsia was detected in Ixodes ricinus ticks feeding on various hosts and in questing
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Phylogenetic analysis revealed a basal position of the novel species in the proximity of R. bellii.
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This is the first report of a basal group rickettsia in Europe.
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‘Candidatus Rickettsia mendelii’ was proposed to designate this microorganism.
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Key words: Rickettsiae, Candidatus Rickettsia mendelii, Ixodes ricinus, Basal group rickettsiae, Ticks,
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Czech Republic
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Abbreviations: CRM - ‘Candidatus Rickettsia mendelii’; ML - maximum likelihood; PP - posterior
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probabilities; REIS - Rickettsia endosymbiont of Ixodes scapularis; SFG - spotted-fever group; TG 2 Page 2 of 19
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Introduction
53 Rickettsiae are obligate intracellular vertebrate bacteria transmitted by blood-feeding arthropod
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vectors (Raoult and Roux, 1997). Rickettsiae are intensely studied not only because of their medical
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importance but also due to the plasticity of their genome related to intracellular lifestyle (Merhej and
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Raoult, 2011). Rickettsiae can be classified into distinct phylogenetic groups; i/ typhi group (TG), ii/
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spotted-fever group (SFG), iii/ basal R. bellii group and iv/ basal R. canadensis group (Fournier and
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Raoult, 2009; Merhej and Raoult, 2011).
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The main vector of tick-borne diseases in Europe is the common tick Ixodes ricinus, which is known to
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carry the SFG agents R. helvetica and R. monacensis, both associated with human cases of
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rickettsiosis (Jado et al., 2007; Nilsson et al., 1999). During examination of I. ricinus ticks feeding on a
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common nightingale (Luscinia megarhynchos), we have detected a novel rickettsial gltA sequence
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showing 84% similarity with the closest taxon R. bellii (Dubska et al., 2012). Subsequently, we found
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this rickettsial DNA in other I. ricinus ticks from various sources. Detailed genetic analysis revealed
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that this novel bacterial sequence pertains to a potentially new rickettsial species. Since the novel
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rickettsial species occurred coincidentally in regions linked with the founder of genetics, Gregor
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Mendel (Matalova, 1999) (Figure 1), we have proposed the name ‘Candidatus Rickettsia mendelii’
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designating
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this
organism.
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Material and Methods
72 Ticks
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Ticks were collected during several tick-borne pathogen surveys performed in the Czech Republic
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from 2005 to 2009. Detailed description of surveys including locations and tick sources is described
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elsewhere (Dubska et al., 2011; Dubska et al., 2009; Dubska et al., 2012). In all surveys, the ticks
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collected in the field were placed in 70% ethanol into individual test tubes and examined
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subsequently in the laboratory. Ticks were classified based on morphological criteria (Nosek and Sixl,
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1972).
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80 Genetic analysis
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Genomic DNA was extracted from ticks by alkaline hydrolysis (Rijpkema et al., 1996). Gene
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amplifications were performed as follows: partial sequences of the 16S rRNA coding gene were
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amplified with fD1 and rP1 primers (Weisburg et al., 1991), the citrate synthase gene (gltA) with
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three different pairs of primers (RpCS.877p and RpCS.1258n, CS-239 and CS-1069, and CS-78 and CS-
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323) (Labruna et al., 2004a; Labruna et al., 2004b; Regnery et al., 1991), the cell surface antigen Sca4
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sequence with two pairs of primers (D1f and D928r; D767f and D1390r) (Sekeyova et al., 2001), the
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17-kDa ricketsial gene (htrA) was amplified with 17k-3 and 17k-5 primers (Labruna et al., 2004a) and
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with 17kD1 and 17kD2 primers (Webb et al., 1990), and the outer membrane protein A (ompA) and B
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(ompB) genes were performed using Rr190.70p and Rr190.602n primers (Regnery et al., 1991), and
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120-M59 and 120-807 primers (Roux and Raoult, 2000), respectively. A section of the gltA gene
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defined by CS-78 and CS-323 primers was directly sequenced in all samples. Additionally, all
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amplicons from samples A-D were cloned into the pGEM®-T Easy Vector (Promega) according to the
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manufacturer's instructions. The ligation reaction was set up using a PCR product:vector molar ratio
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of 3:1. Positive and background controls were prepared in parallel as recommended by the
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manufacturer. The reactions were incubated for 30 minutes on ice, after which tubes were quickly
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centrifuged and 2 µl of ligation mixture were added to 50 µl of XL1 Blue competent cells.
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Transformation of competent cells was performed by heat shock (42°C for 45 seconds) according to
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manufacturer’s instructions. Transformation mixture (100 µl) was plated on LB agar plates containing
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ampicillin, isopropyl β-D-1-thiogalactopyranoside and X-Gal and incubated at 37°C overnight. White
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bacterial colonies were picked and grown overnight at 37°C with agitation in 5 ml Luria broth
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supplemented with ampicillin (100 µg/ml final concentration). Recombinant plasmids were purified
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using the QIAprep® Spin Miniprep kit (Qiagen). At least three clones from each sample were
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sequenced on both strands by a service company (GATC Biotech) and the gltA and 16S rDNA gene
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sequences obtained from different clones were aligned.
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106 Phylogenetic analysis
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Amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA from examined
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organisms were downloaded from GenBank. Datasets were aligned using the multiple sequence
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alignment program MUSCLE (Edgar, 2004) and concatenated in SeaView 4 (Gouy et al., 2010). A total
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of 353 amino acid and 1488 nucleotide positions were included into the final dataset. The alignments
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were checked manually, and gaps in ambiguously aligned regions were excluded from further
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analysis. Genetic similarities among acquired sequences of Candidatus Rickettsia mendelii were
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computed in MEGA 6.06 (Tamura et al., 2013) as pairwise p-distances with pairwise deletion of
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gaps/missing data. Genetic similarities between Candidatus Rickettsia mendelii and other rickettsial
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species were computed in MEGA 6.06 as between group mean p-distances with pairwise deletion of
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gaps/missing data, while all acquired sequences of Candidatus Rickettsia mendelii were pooled into a
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single group and sequences of all other respective species formed their respective groups containing
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a single sequence. Phylogenetic analysis of the concatenated dataset was performed using maximum
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likelihood (ML) and Bayesian interference. Maximum likelihood topology was inferred using the
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gamma corrected General Time Reversible model of evolution (for nucleotides) and WAG (for amino
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acids) as implemented in RAxML 7.4.8 (Stamatakis, 2006). Branching support was estimated from 500
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non-parametric bootstrap replicates using the above-mentioned software and conditions. Only the
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values above 50% are shown. Bayesian posterior probabilities (PP) were estimated using MrBayes
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3.2.2 (Huelsenbeck et al., 2001) employing the models described above for ML analysis. Two
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independent Markov chains were run for 3x10^6 generations of which the first 5x10^5 were omitted
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from topology and PP reconstruction as a burn-in. Only branches with a PP higher than 0.95 were
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considered
be
supported
by
the
model.
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Results
131 Occurrence of ‘Candidatus R. mendelii’
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Candidatus R. mendelii’ was detected in 7 out of 4524 Ixodes ricinus ticks examined during several
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tick-borne pathogen surveys performed in the Czech Republic from 2005 to 2009 (Dubska et al.,
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2011; Dubska et al., 2009; Dubska et al., 2012). A summary of CRM detection during our tick surveys
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is described in Supplementary material 1. Tick species other than I. ricinus found in tick surveys
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included 8 adults of Dermacentor reticulatus from a dog (Literak et al., unpublished data), 2
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subadults of Haemaphysalis concinna (from a dog and a song bird), and 3 nymphs of I. arboricola
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feeding on song birds (Dubska et al., 2011). ‘Candidatus R. mendelii’ (CRM) was detected in both
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subadult (2 nymphs) and adult (1 female, 4 males) I. ricinus ticks acquired from various sources
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including vegetation, birds and a dog (Table 1 and Figure 1).
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Genetic and phylogenetic analysis of the novel rickettsial species
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Samples D-G from a dog were identical in the gltA nucleotide sequence defined by CS-78 and CS-323
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primers. Citrate synthase amino acid sequences acquired from samples B-D with all three pairs of
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primers were mutually more than 99% identical (99.4 – 100%) and were also more than 99% identical
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(99.7 – 100%) to previously published sequences from a tick feeding on the common nightingale
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(sample A) (supplementary material 2A). 16S rDNA sequences from samples A-C are mutually more
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than 99% identical (99.3 – 99.8 %) (supplementary material 2B).
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We found that the ‘Candidatus R. mendelii’ gltA gene is 86.4% identical with R. canadensis, 88% with
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R. monteiroi and 84.2% identical with R. bellii. The studied portion of 16S rDNA was 97.2% identical
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with R. canadensis, 97.5% identical with R. monteiroi and 97.7% identical with R. bellii. OmpA, ompB,
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gene D (sca4), and htrA (17-kDa antigen) were not amplifiable in the investigated isolates, which
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suggested that the novel bacteria does not belong to the SFG or typhi subgroups of ricketsiae
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(Fournier et al., 2003; Raoult et al., 2005). This assumption was further explored employing
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phylogenetic analysis. A maximum likelihood tree of rickettsiae based on comparisons of partial
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amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA genes shows the
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position of ‘Candidatus R. mendelii’ within Rickettsia spp. (Figure 2). Results obtained using a
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concatenated dataset are consistent with those based on single-gene analyses (Supplementary
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material 4). Phylogenetic analysis using Bayesian posterior probabilities confirmed the basal position
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of
isolate,
showing
analogical
topology
(Supplementary
material
4).
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162 Discussion
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Here we report a novel rickettsia species with the proposed name ‘Candidatus Rickettsia mendelii’.
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The novel Rickettsia belongs together with R. bellii, R. monteiroi, and R. canadensis to the most basal
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group of tick-associated rickettsiae. To our knowledge, this is the first report of a basal group
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rickettsia in Europe.
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The highest similarity of ‘Candidatus Rickettsia mendelii‘ to other validated Rickettsia species was
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97.7% with R. bellii according to 16S rDNA sequence and 88.0% with R. monteiroi in the gltA gene.
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Following actual version of consensual guidelines for taxonomic classification of rickettsiae based on
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multiple gene sequencing, organisms with similarity to at least one validated Rickettsia species equal
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or less than 98.1% in rrs (16S) and/or 86.5% in gltA should be considered as a genus other than
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Rickettsia (Fournier and Raoult, 2009). Herein, these cut-off values were determined on sequences of
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20 validated Rickettsia species (Fournier et al., 2003) in the following manner: i/ the similarities in the
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16S rDNA gene ranged from 97.7% between R. prowazekii and R. akari to 99.8% between R. sibirica
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and R. rickettsia, ii/ when R. akari was excluded from the analysis, the cut-off value was recalculated
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to 98.1% and applied to seven unvalidated rickettsial isolates. One of these unvalidated isolates was
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R. heilongjiangensis that was, despite its highest rrs similarity being only 98.0%, consequently
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validated as a novel rickettsiae species (Fournier et al., 2003). Thus, although R. heilongjiangensis did
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not meet the criteria for belonging to the Rickettsia genus, phylogenetic analysis unambiguously
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confirmed R. heilongjiangensis as a rickettsia species (Duan et al., 2014). Similarly, CRM with a 16S
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rDNA similarity of 97.7% did not meet both criteria for Rickettsia spp.; however, phylogenetic
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analysis demonstrated its basal position within the Rickettsia genus. Considering i/ the R.
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heilongjiangensis comparison, ii/ the fact that the phylogenetically closest non-Rickettsia bacterium
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Orientia tsutsugamushi has rrs pairwise nucleotide sequence similarity with 20 validated Rickettsia
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species ranging from 90.2 to 91.0, and finally iii/ the fact that classification study (Fournier et al.,
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2003) included only 2 basal rickettsial species and thus the similarity values were derived mostly
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from SFG group members, there is sufficient evidence to conclude that the sequence-based criteria
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and their cut-off values at the Rickettsia genus level are too strict for the highly diverse non-SFG and
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non-typhus Rickettsia species (Perlman et al., 2006). The Rickettsia genus in its broad sense can
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indeed be divided into distinct clades, i/ “hydra” containing protist-associated Rickettsia, ii/ “torix”
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with Rickettsia from amoeba, leeches and arthropods, iii/ “rhizobius” containing beetle Rickettsia, iv/
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“meloidae” containing Meloidae beetle Rickettsia, v/ “bellii” containing R. bellii and other strains of
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arthropod Rickettsia mostly from insects, and vi/ diverse bacteria containing both arthropod and
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vertebrate Rickettsia that can be further subdivided into the following groups: “onychiurus”,
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“adalia”, “canadensis”, “SFG”, “typhus group”, and finally “transitional group” (Weinert et al., 2009).
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Such expanded recognition of rickettsiae suggests that validated rickettsial species enriched for
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pathogenic bacteria represent only a subset of the Rickettsia genus and that a substantial subset of
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rickettsiae is maintained in the arthropod host population as endosymbionts. Here it is of note that,
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although the majority of symbiotic rickettsiae arthropod hosts are insects, a non-pathogenic
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Rickettsia endosymbiont of Ixodes scapularis (REIS) has been described in I. scapularis (Gillespie et
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al., 2012) which is the American counterpart of I. ricinus. CRM was detected predominantly in male I.
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ricinus ticks and thus transmission of CRM by co-feeding or from a vertebral host is unlikely because
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male ticks do not suck the vertebrate host blood. Thus, the distribution of the novel bacterium is
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suggestive of a transtadial transmission and thus a symbiotic rather than pathogenic ecology of
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‘Candidatus R. mendelii‘.
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Taken together, we propose a new Rickettsia, designated ‘Candidatus Rickettsia mendelii‘, detected
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in Ixodes ricinus from various sources. The new bacterium has a phylogenetic position close to R.
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bellii and further research is required to reveal symbiotic and pathogenic traits of ‘Candidatus R.
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mendelii’
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and
its
host
specificity.
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211 Acknowledgment:
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The authors thank Marek Bednar, Elena Kocianova, Eva Suchanova, and many field coworkers for
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their help in the course of the investigation. The study was funded by CEITEC
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(CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund, and by NPS I
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RECAMO2020 (LO1413) and by ACIU infrastructure (LM2011017), both funded by MEYS.
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Table and figure legend:
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Table 1: Source of ‘Candidatus R. mendelii' samples.
219 Figure 1. Occurrence of ‘Candidatus R. mendelii’ in Czech and Slovak I. ricinus surveys. The Czech
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Republic and Slovakia are highlighted. “A”-“G” indicate ‘Candidatus R. mendelii’ described in this
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study (Table 1). “S”, Putative ‘Candidatus R. mendelii’ gltA DNA detected in a questing tick in
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Bratislava, Slovakia (Spitalska et al., 2014). “N1”, negative finding from Orlicke Zahori. “N2”, negative
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finding from Certak 2005. “M1”, Vrazne, Hyncice, Gregor Mendel’s birth place. “M2”, Brno, where
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Mendel worked when postulating the principles of classical genetics.
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Figure 2. Concatenated phylogenetic tree of rickettsiae based on comparison of the partial amino
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acid sequence of citrate synthase and nucleotide sequence of 16S rDNA. The scale bar represents the
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number of nucleotide changes per site. The gapped branches have been truncated to one half of
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their original length for clarity. Accession numbers of particular sequence used in the alignment can
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be found in supplemental material 3. Members of the spotted fever group (SFG) and typhus group
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(TG)
are
highlighted
in
grey.
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Regnery, R.L., Spruill, C.L., Plikaytis, B.D., 1991. Genotypic identification of rickettsiae and estimation
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of intraspecies sequence divergence for portions of two rickettsial genes. J. Bacteriol. 173, 1576-
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1589.
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Rijpkema, S., Golubic, D., Molkenboer, M., Verbeek-De Kruif, N., Schellekens, J., 1996. Identification
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of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in a Lyme
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borreliosis endemic region of northern Croatia. Exp. Appl. Acarol. 20, 23-30.
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Roux, V., Raoult, D., 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene
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encoding the outer-membrane protein rOmpB (ompB). Int. J. Syst. Evol. Microbiol. 50, 1449-1455.
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Sekeyova, Z., Roux, V., Raoult, D., 2001. Phylogeny of Rickettsia spp. inferred by comparing
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sequences of 'gene D', which encodes an intracytoplasmic protein. Int. J. Syst. Evol. Microbiol. 51,
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1353-1360.
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Spitalska, E., Boldis, V., Derdakova, M., Selyemova, D., Rusnakova Taragelova, V., 2014. Rickettsial
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infection in Ixodes ricinus ticks in urban and natural habitats of Slovakia. Ticks Tick-Borne Dis. 5, 161-
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165.
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Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with
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thousands of taxa and mixed models. Bioinformatics. 22, 2688-2690.
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Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary
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Genetics Analysis Version 6.0. Mol. Biol. Evol. 30, 2725-2729.
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Webb, L., Carl, M., Malloy, D.C., Dasch, G.A., Azad, A.F., 1990. Detection of murine typhus infection in
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fleas by using the polymerase chain reaction. J. Clin. Microbiol. 28, 530-534.
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Weinert, L.A., Werren, J.H., Aebi, A., Stone, G.N., Jiggins, F.M., 2009. Evolution and diversity of
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Rickettsia bacteria. BMC Biol. 7, 6.
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Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal DNA amplification for
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phylogenetic study. J. Bacteriol. 173, 697-703.
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309 310
C D E F
fully-fed nymph male male female male
G
male
Certak, Novy Jicin, 49°34'N, 17°59'E, (Dubska et al., 2009)
Apr 11, 2007
Apr 16, 2006 May 24-29, 2009 May 24-29, 2009 May 30-June 2, 2009 May 30-June 2, 2009
Table 1
Brno, 49°12'N, 16°35'E, (Literak, unpublished data)
M
312
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313 314
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B
common nightingale (Luscinia megarhynchos) European robin (Erithacus rubecula) vegetation flat-coated retriever
us
half-fed nymph
an
311
A
sampling date of location, coordinates, reference CRM-infected tick for the survey and/or description of CRM-infected tick collection site Apr 30, 2008 Mlada Boleslav, 50°25'N 14°54'E, (Dubska et al., 2012)
cr
Sample I. ricinus tick source stage
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S1877-959X(16)30018-8 http://dx.doi.org/doi:10.1016/j.ttbdis.2016.02.004 TTBDIS 610
To appear in: Received date: Revised date: Accepted date:
18-7-2014 10-1-2016 3-2-2016
Please cite this article as: Hajduskova, E., Literak, I., Papousek, I., Costa, F.B., Novakova, M., Labruna, M.B., Zdrazilova-Dubska, L.,‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the Czech Republic, Ticks and Tick-borne Diseases (2016), http://dx.doi.org/10.1016/j.ttbdis.2016.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
‘Candidatus Rickettsia mendelii’, a novel basal group rickettsia detected in Ixodes ricinus ticks in the
2
Czech Republic
3 Eva Hajduskovaa,b, Ivan Literaka,c, Ivo Papouseka, Francisco B. Costad, Marketa Novakovaa,c, Marcelo B.
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Labrunad, Lenka Zdrazilova-Dubska a,e,f (ca)
ip t
4
cr
6 7
a
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of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1/3, 612 42 Brno, Czech Republic
9
b
us
Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University
Institute of Parasitology, Biology Centre, ASCR v.v.i., Branisovska 31, 370 05 Ceske Budejovice,
Czech Republic
11
c
12
Brno, Czech Republic
13
d
14
and Animal Husbandry, University of São Paulo, Avenue Orlando Marques de Paiva, 87 São Paulo,
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Brazil 05508-270
16
e
17
Brno, Czech Republic
18
f
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Brno, Czech Republic
M
CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1/3, 614 42
te
d
Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine
Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53
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an
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Department of Pharmacology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00
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corresponding author: Lenka Zdrazilova-Dubska, Department of Laboratory Medicine, Masaryk
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Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic, +420 543 136 704,
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[email protected].
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24 Abstract:
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A novel rickettsial sequence in the citrate synthase gltA gene indicating a novel Rickettsia species has
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been detected in 7 out of 4524 Ixodes ricinus ticks examined within several surveys performed in the
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Czech Republic from 2005 to 2009. This new Candidatus Rickettsia sp. sequence has been found in 2
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nymphs feeding on wild birds (Luscinia megarhynchos and Erithacus rubecula), in a male tick from
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vegetation, and 4 ticks feeding on a dog (3 males, 1 female tick). Portions of the ompA, ompB, sca4,
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and htrA genes were not amplifiable in these samples. A maximum likelihood tree of rickettsiae
32
based on comparisons of partial amino acid sequences of citrate synthase and nucleotide sequences
33
of 16S rDNA genes and phylogenetic analysis revealed a basal position of the novel species in the
34
proximity of R. bellii and R. canadensis. The novel species has been named ‘Candidatus Rickettsia
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mendelii’ after the founder of genetics, Gregor Mendel.
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Highlights: •
40 41
ticks.
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A novel rickettsia was detected in Ixodes ricinus ticks feeding on various hosts and in questing
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•
Phylogenetic analysis revealed a basal position of the novel species in the proximity of R. bellii.
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•
This is the first report of a basal group rickettsia in Europe.
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•
‘Candidatus Rickettsia mendelii’ was proposed to designate this microorganism.
44 45
Key words: Rickettsiae, Candidatus Rickettsia mendelii, Ixodes ricinus, Basal group rickettsiae, Ticks,
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Czech Republic
47 48
Abbreviations: CRM - ‘Candidatus Rickettsia mendelii’; ML - maximum likelihood; PP - posterior
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probabilities; REIS - Rickettsia endosymbiont of Ixodes scapularis; SFG - spotted-fever group; TG 2 Page 2 of 19
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51 52
Introduction
53 Rickettsiae are obligate intracellular vertebrate bacteria transmitted by blood-feeding arthropod
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vectors (Raoult and Roux, 1997). Rickettsiae are intensely studied not only because of their medical
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importance but also due to the plasticity of their genome related to intracellular lifestyle (Merhej and
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Raoult, 2011). Rickettsiae can be classified into distinct phylogenetic groups; i/ typhi group (TG), ii/
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spotted-fever group (SFG), iii/ basal R. bellii group and iv/ basal R. canadensis group (Fournier and
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Raoult, 2009; Merhej and Raoult, 2011).
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The main vector of tick-borne diseases in Europe is the common tick Ixodes ricinus, which is known to
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carry the SFG agents R. helvetica and R. monacensis, both associated with human cases of
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rickettsiosis (Jado et al., 2007; Nilsson et al., 1999). During examination of I. ricinus ticks feeding on a
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common nightingale (Luscinia megarhynchos), we have detected a novel rickettsial gltA sequence
64
showing 84% similarity with the closest taxon R. bellii (Dubska et al., 2012). Subsequently, we found
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this rickettsial DNA in other I. ricinus ticks from various sources. Detailed genetic analysis revealed
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that this novel bacterial sequence pertains to a potentially new rickettsial species. Since the novel
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rickettsial species occurred coincidentally in regions linked with the founder of genetics, Gregor
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Mendel (Matalova, 1999) (Figure 1), we have proposed the name ‘Candidatus Rickettsia mendelii’
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designating
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this
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Material and Methods
72 Ticks
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Ticks were collected during several tick-borne pathogen surveys performed in the Czech Republic
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from 2005 to 2009. Detailed description of surveys including locations and tick sources is described
76
elsewhere (Dubska et al., 2011; Dubska et al., 2009; Dubska et al., 2012). In all surveys, the ticks
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collected in the field were placed in 70% ethanol into individual test tubes and examined
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subsequently in the laboratory. Ticks were classified based on morphological criteria (Nosek and Sixl,
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1972).
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80 Genetic analysis
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Genomic DNA was extracted from ticks by alkaline hydrolysis (Rijpkema et al., 1996). Gene
83
amplifications were performed as follows: partial sequences of the 16S rRNA coding gene were
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amplified with fD1 and rP1 primers (Weisburg et al., 1991), the citrate synthase gene (gltA) with
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three different pairs of primers (RpCS.877p and RpCS.1258n, CS-239 and CS-1069, and CS-78 and CS-
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323) (Labruna et al., 2004a; Labruna et al., 2004b; Regnery et al., 1991), the cell surface antigen Sca4
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sequence with two pairs of primers (D1f and D928r; D767f and D1390r) (Sekeyova et al., 2001), the
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17-kDa ricketsial gene (htrA) was amplified with 17k-3 and 17k-5 primers (Labruna et al., 2004a) and
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with 17kD1 and 17kD2 primers (Webb et al., 1990), and the outer membrane protein A (ompA) and B
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(ompB) genes were performed using Rr190.70p and Rr190.602n primers (Regnery et al., 1991), and
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120-M59 and 120-807 primers (Roux and Raoult, 2000), respectively. A section of the gltA gene
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defined by CS-78 and CS-323 primers was directly sequenced in all samples. Additionally, all
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amplicons from samples A-D were cloned into the pGEM®-T Easy Vector (Promega) according to the
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manufacturer's instructions. The ligation reaction was set up using a PCR product:vector molar ratio
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of 3:1. Positive and background controls were prepared in parallel as recommended by the
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manufacturer. The reactions were incubated for 30 minutes on ice, after which tubes were quickly
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centrifuged and 2 µl of ligation mixture were added to 50 µl of XL1 Blue competent cells.
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Transformation of competent cells was performed by heat shock (42°C for 45 seconds) according to
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manufacturer’s instructions. Transformation mixture (100 µl) was plated on LB agar plates containing
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ampicillin, isopropyl β-D-1-thiogalactopyranoside and X-Gal and incubated at 37°C overnight. White
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bacterial colonies were picked and grown overnight at 37°C with agitation in 5 ml Luria broth
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supplemented with ampicillin (100 µg/ml final concentration). Recombinant plasmids were purified
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using the QIAprep® Spin Miniprep kit (Qiagen). At least three clones from each sample were
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sequenced on both strands by a service company (GATC Biotech) and the gltA and 16S rDNA gene
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sequences obtained from different clones were aligned.
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106 Phylogenetic analysis
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Amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA from examined
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organisms were downloaded from GenBank. Datasets were aligned using the multiple sequence
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alignment program MUSCLE (Edgar, 2004) and concatenated in SeaView 4 (Gouy et al., 2010). A total
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of 353 amino acid and 1488 nucleotide positions were included into the final dataset. The alignments
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were checked manually, and gaps in ambiguously aligned regions were excluded from further
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analysis. Genetic similarities among acquired sequences of Candidatus Rickettsia mendelii were
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computed in MEGA 6.06 (Tamura et al., 2013) as pairwise p-distances with pairwise deletion of
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gaps/missing data. Genetic similarities between Candidatus Rickettsia mendelii and other rickettsial
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species were computed in MEGA 6.06 as between group mean p-distances with pairwise deletion of
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gaps/missing data, while all acquired sequences of Candidatus Rickettsia mendelii were pooled into a
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single group and sequences of all other respective species formed their respective groups containing
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a single sequence. Phylogenetic analysis of the concatenated dataset was performed using maximum
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likelihood (ML) and Bayesian interference. Maximum likelihood topology was inferred using the
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gamma corrected General Time Reversible model of evolution (for nucleotides) and WAG (for amino
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acids) as implemented in RAxML 7.4.8 (Stamatakis, 2006). Branching support was estimated from 500
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non-parametric bootstrap replicates using the above-mentioned software and conditions. Only the
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values above 50% are shown. Bayesian posterior probabilities (PP) were estimated using MrBayes
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3.2.2 (Huelsenbeck et al., 2001) employing the models described above for ML analysis. Two
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independent Markov chains were run for 3x10^6 generations of which the first 5x10^5 were omitted
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from topology and PP reconstruction as a burn-in. Only branches with a PP higher than 0.95 were
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considered
be
supported
by
the
model.
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129 130
Results
131 Occurrence of ‘Candidatus R. mendelii’
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Candidatus R. mendelii’ was detected in 7 out of 4524 Ixodes ricinus ticks examined during several
134
tick-borne pathogen surveys performed in the Czech Republic from 2005 to 2009 (Dubska et al.,
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2011; Dubska et al., 2009; Dubska et al., 2012). A summary of CRM detection during our tick surveys
136
is described in Supplementary material 1. Tick species other than I. ricinus found in tick surveys
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included 8 adults of Dermacentor reticulatus from a dog (Literak et al., unpublished data), 2
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subadults of Haemaphysalis concinna (from a dog and a song bird), and 3 nymphs of I. arboricola
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feeding on song birds (Dubska et al., 2011). ‘Candidatus R. mendelii’ (CRM) was detected in both
140
subadult (2 nymphs) and adult (1 female, 4 males) I. ricinus ticks acquired from various sources
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including vegetation, birds and a dog (Table 1 and Figure 1).
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Genetic and phylogenetic analysis of the novel rickettsial species
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Samples D-G from a dog were identical in the gltA nucleotide sequence defined by CS-78 and CS-323
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primers. Citrate synthase amino acid sequences acquired from samples B-D with all three pairs of
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primers were mutually more than 99% identical (99.4 – 100%) and were also more than 99% identical
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(99.7 – 100%) to previously published sequences from a tick feeding on the common nightingale
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(sample A) (supplementary material 2A). 16S rDNA sequences from samples A-C are mutually more
149
than 99% identical (99.3 – 99.8 %) (supplementary material 2B).
150
We found that the ‘Candidatus R. mendelii’ gltA gene is 86.4% identical with R. canadensis, 88% with
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R. monteiroi and 84.2% identical with R. bellii. The studied portion of 16S rDNA was 97.2% identical
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with R. canadensis, 97.5% identical with R. monteiroi and 97.7% identical with R. bellii. OmpA, ompB,
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gene D (sca4), and htrA (17-kDa antigen) were not amplifiable in the investigated isolates, which
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suggested that the novel bacteria does not belong to the SFG or typhi subgroups of ricketsiae
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(Fournier et al., 2003; Raoult et al., 2005). This assumption was further explored employing
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phylogenetic analysis. A maximum likelihood tree of rickettsiae based on comparisons of partial
157
amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA genes shows the
158
position of ‘Candidatus R. mendelii’ within Rickettsia spp. (Figure 2). Results obtained using a
159
concatenated dataset are consistent with those based on single-gene analyses (Supplementary
160
material 4). Phylogenetic analysis using Bayesian posterior probabilities confirmed the basal position
161
of
isolate,
showing
analogical
topology
(Supplementary
material
4).
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162 Discussion
164
Here we report a novel rickettsia species with the proposed name ‘Candidatus Rickettsia mendelii’.
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The novel Rickettsia belongs together with R. bellii, R. monteiroi, and R. canadensis to the most basal
166
group of tick-associated rickettsiae. To our knowledge, this is the first report of a basal group
167
rickettsia in Europe.
168
The highest similarity of ‘Candidatus Rickettsia mendelii‘ to other validated Rickettsia species was
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97.7% with R. bellii according to 16S rDNA sequence and 88.0% with R. monteiroi in the gltA gene.
170
Following actual version of consensual guidelines for taxonomic classification of rickettsiae based on
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multiple gene sequencing, organisms with similarity to at least one validated Rickettsia species equal
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or less than 98.1% in rrs (16S) and/or 86.5% in gltA should be considered as a genus other than
173
Rickettsia (Fournier and Raoult, 2009). Herein, these cut-off values were determined on sequences of
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20 validated Rickettsia species (Fournier et al., 2003) in the following manner: i/ the similarities in the
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16S rDNA gene ranged from 97.7% between R. prowazekii and R. akari to 99.8% between R. sibirica
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and R. rickettsia, ii/ when R. akari was excluded from the analysis, the cut-off value was recalculated
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to 98.1% and applied to seven unvalidated rickettsial isolates. One of these unvalidated isolates was
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R. heilongjiangensis that was, despite its highest rrs similarity being only 98.0%, consequently
179
validated as a novel rickettsiae species (Fournier et al., 2003). Thus, although R. heilongjiangensis did
180
not meet the criteria for belonging to the Rickettsia genus, phylogenetic analysis unambiguously
181
confirmed R. heilongjiangensis as a rickettsia species (Duan et al., 2014). Similarly, CRM with a 16S
182
rDNA similarity of 97.7% did not meet both criteria for Rickettsia spp.; however, phylogenetic
183
analysis demonstrated its basal position within the Rickettsia genus. Considering i/ the R.
184
heilongjiangensis comparison, ii/ the fact that the phylogenetically closest non-Rickettsia bacterium
185
Orientia tsutsugamushi has rrs pairwise nucleotide sequence similarity with 20 validated Rickettsia
186
species ranging from 90.2 to 91.0, and finally iii/ the fact that classification study (Fournier et al.,
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2003) included only 2 basal rickettsial species and thus the similarity values were derived mostly
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from SFG group members, there is sufficient evidence to conclude that the sequence-based criteria
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and their cut-off values at the Rickettsia genus level are too strict for the highly diverse non-SFG and
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non-typhus Rickettsia species (Perlman et al., 2006). The Rickettsia genus in its broad sense can
191
indeed be divided into distinct clades, i/ “hydra” containing protist-associated Rickettsia, ii/ “torix”
192
with Rickettsia from amoeba, leeches and arthropods, iii/ “rhizobius” containing beetle Rickettsia, iv/
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“meloidae” containing Meloidae beetle Rickettsia, v/ “bellii” containing R. bellii and other strains of
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arthropod Rickettsia mostly from insects, and vi/ diverse bacteria containing both arthropod and
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vertebrate Rickettsia that can be further subdivided into the following groups: “onychiurus”,
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“adalia”, “canadensis”, “SFG”, “typhus group”, and finally “transitional group” (Weinert et al., 2009).
197
Such expanded recognition of rickettsiae suggests that validated rickettsial species enriched for
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pathogenic bacteria represent only a subset of the Rickettsia genus and that a substantial subset of
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rickettsiae is maintained in the arthropod host population as endosymbionts. Here it is of note that,
200
although the majority of symbiotic rickettsiae arthropod hosts are insects, a non-pathogenic
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Rickettsia endosymbiont of Ixodes scapularis (REIS) has been described in I. scapularis (Gillespie et
202
al., 2012) which is the American counterpart of I. ricinus. CRM was detected predominantly in male I.
203
ricinus ticks and thus transmission of CRM by co-feeding or from a vertebral host is unlikely because
204
male ticks do not suck the vertebrate host blood. Thus, the distribution of the novel bacterium is
205
suggestive of a transtadial transmission and thus a symbiotic rather than pathogenic ecology of
206
‘Candidatus R. mendelii‘.
207
Taken together, we propose a new Rickettsia, designated ‘Candidatus Rickettsia mendelii‘, detected
208
in Ixodes ricinus from various sources. The new bacterium has a phylogenetic position close to R.
209
bellii and further research is required to reveal symbiotic and pathogenic traits of ‘Candidatus R.
210
mendelii’
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and
its
host
specificity.
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211 Acknowledgment:
213
The authors thank Marek Bednar, Elena Kocianova, Eva Suchanova, and many field coworkers for
214
their help in the course of the investigation. The study was funded by CEITEC
215
(CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund, and by NPS I
216
RECAMO2020 (LO1413) and by ACIU infrastructure (LM2011017), both funded by MEYS.
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Table and figure legend:
218
Table 1: Source of ‘Candidatus R. mendelii' samples.
219 Figure 1. Occurrence of ‘Candidatus R. mendelii’ in Czech and Slovak I. ricinus surveys. The Czech
221
Republic and Slovakia are highlighted. “A”-“G” indicate ‘Candidatus R. mendelii’ described in this
222
study (Table 1). “S”, Putative ‘Candidatus R. mendelii’ gltA DNA detected in a questing tick in
223
Bratislava, Slovakia (Spitalska et al., 2014). “N1”, negative finding from Orlicke Zahori. “N2”, negative
224
finding from Certak 2005. “M1”, Vrazne, Hyncice, Gregor Mendel’s birth place. “M2”, Brno, where
225
Mendel worked when postulating the principles of classical genetics.
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an
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Figure 2. Concatenated phylogenetic tree of rickettsiae based on comparison of the partial amino
228
acid sequence of citrate synthase and nucleotide sequence of 16S rDNA. The scale bar represents the
229
number of nucleotide changes per site. The gapped branches have been truncated to one half of
230
their original length for clarity. Accession numbers of particular sequence used in the alignment can
231
be found in supplemental material 3. Members of the spotted fever group (SFG) and typhus group
232
(TG)
are
highlighted
in
grey.
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233 References:
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Duan, C., Xiong, X., Qi, Y., Gong, W., Jiao, J., Wen, B., 2014. Genomic and comparative genomic
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Gillespie, J.J., Joardar, V., Williams, K.P., Driscoll, T., Hostetler, J.B., Nordberg, E., Shukla, M., Walenz,
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309 310
C D E F
fully-fed nymph male male female male
G
male
Certak, Novy Jicin, 49°34'N, 17°59'E, (Dubska et al., 2009)
Apr 11, 2007
Apr 16, 2006 May 24-29, 2009 May 24-29, 2009 May 30-June 2, 2009 May 30-June 2, 2009
Table 1
Brno, 49°12'N, 16°35'E, (Literak, unpublished data)
M
312
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313 314
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B
common nightingale (Luscinia megarhynchos) European robin (Erithacus rubecula) vegetation flat-coated retriever
us
half-fed nymph
an
311
A
sampling date of location, coordinates, reference CRM-infected tick for the survey and/or description of CRM-infected tick collection site Apr 30, 2008 Mlada Boleslav, 50°25'N 14°54'E, (Dubska et al., 2012)
cr
Sample I. ricinus tick source stage
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