Mohamed Rahal

Hacene Medkour

Adama Zan Diarra

Idir Bitam

Philippe Parola

Oleg Mediannikov

Mephi

Molecular identification and evaluation of Coxiella-like endosymbionts genetic diversity carried by cattle ticks in Algeria

Keywords: Coxiella-like, Ticks, Cattle, qPCR, sequencing, Algeria

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Introduction

Ticks and mosquitoes are considered the most important vectors of pathogenic microorganisms for both humans and terrestrial vertebrate animals in the world [START_REF] Dehhaghi | Human tick-borne diseases in Australia[END_REF]. A high number of infectious agents, such as viruses, bacteria and protozoans, are transmitted by ticks [START_REF] Brinkmann | A crosssectional screening by next-generation sequencing reveals Rickettsia , Theileria and Hemolivia species in ticks from Anatolia[END_REF].

All tick-borne diseases (TBDs) might be considered zoonotic diseases that represent one of the most rapidly expanding arthropod-borne diseases [START_REF] Pfäffle | The ecology of tick-borne diseases[END_REF], since several tickborne infections with a significant impact on human and animal's health have been observed in recent decades [START_REF] Brinkmann | A crosssectional screening by next-generation sequencing reveals Rickettsia , Theileria and Hemolivia species in ticks from Anatolia[END_REF].

To date, about 900 tick species have been identified and classified worldwide, while in Africa, 223 tick species have been described, 180 of which are classified as hard and 43 as soft ticks [START_REF] Diarra | Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali[END_REF]. Besides, the pathogens carried by these ticks, diverse commensal and symbiotic microorganisms are also present, such as Coxiella-like endosymbionts (CLEs) [START_REF] Špitalská | Diversity of Coxiella-like and Francisella-like endosymbionts, and Rickettsia spp., Coxiella burnetii as pathogens in the tick populations of Slovakia, Central Europe[END_REF][START_REF] Zhong | Coxiella-like endosymbionts[END_REF].

Coxiella burnetii, the Q fever agent distributed worldwide, has already been reported many times in Algeria [START_REF] Angelakis | Coxiella burnetiipositive PCR in febrile patients in rural and urban Africa[END_REF][START_REF] Khaled | Serological and molecular evidence of Q fever among small ruminant flocks in Algeria[END_REF]. Multispacer type 20 was determined to be the circulating genotype in cattle in the northern Algeria [START_REF] Rahal | Genotyping of Coxiella burnetii detected in placental tissues from aborted dairy cattle in the north of Algeria[END_REF].

According to [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF], all strains of C. burnetii are the descendants of a Coxiella-like progenitor. Consequently, they originated from this group hosted by ticks. No strains of Coxiella-like are yet cultured in the environment of vertebrate cells, suggesting that their metabolic needs are different from those of C. burnetii. In addition, the recent evolvement of C. burnetii from an inherited symbiont of ticks allowed it to infect vertebrates after acquisitions of novel virulence factors [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF].

According to the recent sequencing of Coxiella-like bacterium found in Amblyomma americanum [START_REF] Smith | A Coxiella-like endosymbiont is a potential vitamin source for the lone star tick[END_REF], recognizable virulence genes have not been found and the main biosynthesis pathways of vitamins and cofactors are encoded by its genome. Thus, it may be a vitamin-provisioning endosymbiont [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF]. Such bacterial functions might be involved in a mutualistic interaction with the tick host by compensating nutritional deficiencies.

In fact [START_REF] Guizzo | A Coxiella mutualist symbiont is essential to the development of Rhipicephalus microplus[END_REF] and [START_REF] Gottlieb | Distinctive genome reduction rates revealed by genomic analyses of two Coxiella-like endosymbionts in ticks[END_REF] reported that the biosynthetic pathways for the vitamins Biotin (B7), Riboflavin (B2), Pyridoxine (B6), Folic acid (B9) and Pantothenate (B5) are found in all CLE. In addition, the coding genes for the cofactors Flavin adenine dinucleotide (FAD) and Coenzyme A (CoA) are found in all CLEs A hypothesis is supported with the observed decreases in ticks fecundity and life-history traits successive to the symbiont elimination [START_REF] René-Martellet | Bacterial microbiota associated with Rhipicephalus sanguineus (s.l.) ticks from France, Senegal and Arizona[END_REF]. The study conducted by [START_REF] Guizzo | A Coxiella mutualist symbiont is essential to the development of Rhipicephalus microplus[END_REF] and [START_REF] Zhong | Antibiotic treatment of the tick vector Amblyomma americanum reduced reproductive fitness[END_REF] showed that tetracycline treatment of ticks led to the reduction of levels of bacteria in progeny in 74 % for eggs and 90 % for larvae, and hampered the tick to reach the adult life stage, as no tick was able to progress beyond the metanymph stage. While [START_REF] Lalzar | Composition and seasonal variation of Rhipicephalus turanicus and Rhipicephalus sanguineus bacterial communities[END_REF], suggested vertical transmission with a high fidelity of CLE since they identified it in eggs and larvae of Rhipicephalus sanguineus sensu lato-associated (R.

sanguineus SL-A). This vertical transmission showed 98 % of the 16S rRNA sequence identity in both eggs and larvae [START_REF] Guizzo | A Coxiella mutualist symbiont is essential to the development of Rhipicephalus microplus[END_REF], which may explain the high frequency of Coxiella-like in the tick populations.

However, Coxiella-like was confirmed to be a new agent of human infections. These bacteria can possibly lead to an atypical scalp eschar and neck lymphadenopathy syndrome with delayed evolution to crust eschar in the area of the tick-bite [START_REF] Guimard | Case report: Scalp eschar and neck lymphadenopathy associated with bacteremia due to Coxiella-like bacteria[END_REF].

A genetic variant of R. sanguineus SL-A Coxiella-like bacterium was detected in skin biopsy samples and ticks collected from patients with eschar [START_REF] Angelakis | Candidatus Coxiella massiliensis infection[END_REF]. This is evidence that at least this strain might be responsible for scalp eschar and neck lymphadenopathy after a tick bite [START_REF] Guimard | Case report: Scalp eschar and neck lymphadenopathy associated with bacteremia due to Coxiella-like bacteria[END_REF]. Furthermore, a multiorgan infection with a Coxiella-like organism was determined to be the cause of death of a female eclectus parrot Eclectus roratus [START_REF] Vapniarsky | Systemic Coxiella -like infection with Myocarditis and Hepatitis in an Eclectus Parrot (Eclectus roratus)[END_REF].

Although Coxiella-like bacteria and C. burnetii share a certain degree of identity, it is not high enough to be clustered in the same species [START_REF] Angelakis | Candidatus Coxiella massiliensis infection[END_REF]. All Coxiella-like bacteria undoubtfully belong to the genus Coxiella in the gamma-subgroup of the Proteobacteria. This taxonomy was based on the 16S rRNA nucleotide sequences. The sequence identity of the 16S rRNA gene among the Coxiella -like bacteria varies from species to species. The assessment of 16S rRNA nucleotide sequencing between Coxiella-like strains showed that the maximal identity between them reached 98 %, while the minimal declined to 91 % [START_REF] Zhong | Coxiella-like endosymbionts[END_REF].

The aim of this study was to assess the occurrence and genetic diversity of Coxiella-like ticks in cattle in Algeria, where no studies on Coxiella-like have been conducted to date.

Material and Methods

Period and area of the study:

Seven hundred sixty-five (765) ticks were collected from dairy cattle between 2013 and 2015, mainly in spring and summer. All ticks (engorged and non-engorged) were collected manually with forceps, as described before [START_REF] Diarra | Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali[END_REF]. The ticks were counted, pooled in tubes and kept frozen at -20 C until morphological, molecular and MALDI-TOF MS analyses. Samples were transported from Algeria to the IHU-Méditerranée Infection, Marseille, France to perform analysis.

The samples collection was carried out in three provinces in northern Algeria, two of which were in the central northern region named Blida (36° 33′3.258″N, 2° 54′31.113″E) and Medea (36° 15′43.54″N, 2° 45′18.947″E), and the third location is Batna (35° 39′ 34.671″N, 6° 19′42.839″E) located in northeastern of Algeria (Fig. 2).

In Blida, 469 ticks were collected, 238 of which were collected from cattle living in the wild in the mountains and brought to the farm only once a month by the farmer. The climate in Blida province is Mediterranean. In winter, precipitation is more abundant than in summer, with about 791 millimeters of precipitation per year. The average annual temperature in Blida is 17.9 °C. However, in Medea (130 ticks), the climate is distinguished by its position above the Tellian Atlas mountains, its altitude of 1,240 meters and its exposed to winds and waves of westerly currents, Blida and Medea are characterized by Mediterranean climate with dry and hot summers [START_REF] Kottek | World map of the Köppen-Geiger climate classification updated[END_REF]. While Batna is located in the Aurès Mountains (part of the Atlas Mountains), at 1,048 meters above sea level. Batna (166 ticks) has a cold steppe climate [START_REF] Kottek | World map of the Köppen-Geiger climate classification updated[END_REF], with Mediterranean influences and average annual precipitation of 326 mm. Summers are moderately hot (by Saharan standards) and dry. Winters are chilly and wetter, with the possibility of snowfall, more details about the long-term mean temperature of the coldest and the warmest month are shown in the Fig. 1.

Morphological identification

This task was performed down to species level if possible, while engorged ticks were identified at the genus level only. The tick identification was performed twice by two expert tick entomologists and checked using previously established taxonomic identification keys [START_REF] Walker | Ticks of domestic animals in africa : a guide to identification of species[END_REF]. This task was performed under a microscope at a magnification of ×56 (Zeiss Axio Zoom.V16, Zeiss, Marly le Roi, France).

DNA extraction

To complete the molecular identification of ticks and the screening for the presence of Coxiella-like bacteria, 147 of the 735 ticks were selected according to their previous morphological identification. The selection was accomplished randomly conferring to their diversities. Thus the selection concerned all species identified morphologically (15 ticks maximum for each specie, in case we had low number of species type e.g. Ixodes ricinus 5/735 all of them were chosen). Moreover the selected ticks belonged to both sexes (male and female), two life stages (nymph, adult), and different engorgement states (engorged or unfed).

DNA extraction was carried out according to the manufacturer's instructions. Briefly, each half tick without legs was incubated at 56°C overnight with 180 μL of G2 lysis buffer (Qiagen, Hilden, Germany) and 20 μL proteinase K (Hilden, Germany). Successively, the extraction using an EZ1 DNA Tissue Kit (Hilden, Germany) and EZ1 BioRobot extraction device was realized. Finally, 200 μL total genomic DNA was eluted in Tris-EDTA (TE) buffer (Hilden, Germany) and kept at -20 °C until use.

Coxiella-like qPCR screening

All selected ticks (20 %; 147/735) were screened for the presence of Coxiella-like endosymbionts, using a qPCR assay targeting the rrs gene [START_REF] Angelakis | Candidatus Coxiella massiliensis infection[END_REF]. Reaction mix for qPCR assay contained 5 μl of the DNA template, 10 μl of Eurogentec Master Mix Roche, 0.5 μl UDG, 0.5 μl of each primer (at 20 µM concentration), and 0.5 μl of the FAMlabeled probe (at 5 µM concentration), (Table 1) and 3 μl of distilled water DNAse and RNAse free, for a final volume of 20 μl. The qPCR amplification was performed in a CFX96 Real-Time system (Bio-rad Laboratories, Foster City, CA, USA). with the following thermal profile: the first step consisted in an incubation at 50°C for two min and an initial denaturation step at 95°C for five min, followed by 40 cycles of denaturation at 95°C for 5 s and annealingextension at 60°C for 30 s. We included master mixture in two wells as negative controls for each test, while DNA from Rhipicephalus bursa reared in the laboratory, was used as positive control. Samples were considered positive when the cycle threshold (Ct) was lower than 35 Ct.

Molecular identification of ticks by 16S rRNA gene sequencing

The morphological identification of ticks showed positive results during the screening (76 ticks) was confirmed by 16S rRNA gene sequencing for 58/76 of them. All selected samples were subjected to standard PCR in an automated DNA thermal cycler. Details about the systems used in the standard PCR are summarized in Table 1.

The standard PCR assays consisted of a volume of 25 µl, including 12.5 µl of Amplitaq Gold Master Mix, 0.75 µl of each primer (at 20 µM concentration), 5 μl of DNA model and water. The thermal cycling conditions were: incubation step at 95°C for 15 min, 40 cycles of 1 min at 95°C, 30 s annealing at hybridization temperature and one min at 72°C followed by a final extension for five min at 72°C (Table 1). PCR amplification was performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). The results of amplification were visualized by electrophoresis on 2 % agarose gel. The purification of PCR products was performed using Nucleo Fast 96 PCR plates (Macherey Nagel EURL, Hoerdt, France) according to the manufacturer's instructions. The amplicons were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems). The sequences obtained were assembled and analyzed using the ChromasPro software (version 1.34) (Technelysium Pty. Ltd., Tewantin, Australia). Finally, assembled sequences were compared with those available in GenBank using the BLAST server from the National Centre for Biotechnology (http://www.ncbi.nlm.nih.gov/blast/).

Coxiella-like typing

Following the precise molecular identification of ticks, two strains samples belonging to the same species were selected for the typing by PCR / sequencing of the Coxiella-like 16S rRNA and GroEL partial genes, since Coxiella-like bacteria are hypothetically specific endosymbionts of ticks according to the species [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF].

The experimental protocol for the conventional PCRs, purification of PCR products, sequencing, assembly and sequences analysis were performed as described above. These tasks were repeated four times for each system (GroEL and 16S) to support the finding and avoid all possibility of contamination between samples.

Phylogenetic analyses

To carry out the construction of the phylogenetic tree, the Coxiella-like obtained sequences in the present study were aligned with reference sequences available in the GenBank database using ClustalW multiple sequence alignment algorithm. A maximum-likelihood method (Kimura 2-parameter model) was used to infer the phylogenetic analyses and tree reconstruction was performed using MEGA6.06 software [START_REF] Tamura | MEGA6: Molecular evolutionary genetics analysis version 6.0[END_REF]. Bootstrap analyses were conducted using 500 replicates.

Statistical analysis

After set of databases Microsoft Excel® program (Microsoft Corp. Redmont, USA), a descriptive study of ticks' population and of the CLE carriage was performed. The statistical analysis was conducted using XLSTAT Addinsoft version 2018.7 (Data Analysis and Statistical Solution for Microsoft Excel, Paris, France). The comparison of CLE infection's prevalence according to the regions was accomplished using Khi-2 test, for that statistical signification was considered at P-value < 0.05.

Results

Tick identification

In this study, 735 hard ticks were collected from cattle. Thirteen species belonging to five genera were identified as cattle ticks in the three northern provinces of Algeria. Almost all of ticks in north-central Algeria (Blida and Medea) belonged to the genus Rhipicephalus, while in Batna, in the northeast, species belonging to genus Hyalomma were the most often identified ticks. More details are provided in Fig. 2.

Prevalence of Coxiella-like in ticks collected in northern of Algeria

In this study, we performed an extensive screening of Coxiella-like on 147 selected ticks (20 % of all ticks collected were selected for DNA extraction). Using qPCR, the prevalence revealed in this study was 51.7 % (76/147) among all the selected ticks for the screening.

Phylogenetic analysis

Sequencing of the positive samples using the Coxiella-like specific primers 16S rRNA failed in 10 of 23 samples, as a result 13 samples were successfully sequenced, and provided a BLAST identity percentage greater than 98 % for better identification (Table 2).

Sequencing using the specific GroEL primers also failed in 19/23 sequenced with these primers, so only four could be sequenced. 4).

Discussion

This study is to the best of our knowledge the first report on the occurrence and diversity of Coxiella-like in cattle ticks from Algeria. The study highlighted the presence of two genera of ticks that constitute the dominant populations of ticks infesting livestock in northern Algeria.

In Blida and Medea, Rhipicephalus spp. are dominant, especially, R. bursa and R. sanguineus SL-A (Fig. 2). R. bursa is a tick of the Mediterranean climatic region, which is the characteristic climate of the concerned provinces. In Africa, it is confined to the coastal areas from Morocco to Libya including Algeria [START_REF] Walker | Ticks of domestic animals in africa : a guide to identification of species[END_REF]. R. sanguineus SL-A, is specialized to domestic dogs, but it may be found on cattle [START_REF] Walker | Ticks of domestic animals in africa : a guide to identification of species[END_REF].

These details support our findings. In Batna, northeastern Algeria, the dominant tick genus was Hyalomma especially H. excavatum. This species is adapted to the Mediterranean and steppe climatic regions of North Africa and to steppe climatic regions elsewhere in its wide range [START_REF] Walker | Ticks of domestic animals in africa : a guide to identification of species[END_REF].

For the first time in Algeria, we report the occurrence of Coxiella-like in ixodid ticks. The prevalence was 51.7 % (76/147) among all the selected ticks for the screening. Coxiella-like DNA was detected in all tick species identified in the present study with different percentages.

The highest prevalence was reported in the genus Rhipicephalus 70 % (53 out of 75), while it was lower in other genera such as Hyalomma with 37 % (26 out of 70). This prevalence in Algeria seems to be lower than those reported worldwide. Actually, [START_REF] Guizzo | A Coxiella mutualist symbiont is essential to the development of Rhipicephalus microplus[END_REF] reported that the treatment of cattle with tetracycline (frequently used in Algeria), leads to the reduction of bacteria levels in the progeny of ticks. These facts might explain the low prevalence reported in this study.

In the study conducted by [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF] Coxiella-like was detected in >90 % of tested ticks, which is extremely high. The screening was performed in 916 tick specimens from 58 species belonging to the two main tick families, Ixodidae (36 species) and Argasidae ( 22species). Coxiella was found in most of the tested genera of hard ticks (Rhipicephalus, Ixodes, Amblyomma, Dermacentor, Haemaphysalis) and soft ticks (Ornithodoros, Argas).

Actually, the prevalence of Coxiella-like varies widely in different tick species. In Europe and North America, it rates from 6.25 % (3/48) in R. sanguineus SL-A to 100 % (50/50) in A.

americanum [START_REF] Zhong | Coxiella-like endosymbionts[END_REF]. [START_REF] Machado-Ferreira | Coxiella symbionts are widespread into hard ticks[END_REF] reported that Coxiella DNA was detected in 37 % (10/27) of Rhipicephalus microplus (5/15 adult females, 4/11 adult males and 1/1 nymph), and in 61 % (29/47) of the R. sanguineus SL-A (15/24 adult females 13/18 adult males 1/5 nymph). These details support our findings in Algeria where the prevalence ranged from 14 % (4/28) for H. marginatum to 81 % (26/32) for R. bursa, to 2/2 for D. marginatus.

In Tunisia, [START_REF] Selmi | Molecular detection and genetic characterization of the potentially pathogenic Coxiella burnetii and the endosymbiotic Candidatus Midichloria mitochondrii in ticks infesting camels (Camelus dromedarius) from Tunisia[END_REF] reported that Coxiella-like DNA was detected in 10 ± 0.04 % (16/158) of Hyalomma impeltatum and 6% (10/160) of Hyalomma dromedarii (partially engorged adult ticks) with an overall prevalence of 8 % (26/327). This is close to our results

regarding Hyalomma genus with 37.1 %. [START_REF] Khoo | Coxiella detection in ticks from wildlife and livestock in Malaysia[END_REF] but also on the bioclimatic area. Our results report a higher prevalence in Blida and Medea, characterized by Mediterranean climate, than in Batna with a cold semi-arid climate. In addition, in Malaysia, which is characterized by an equatorial climate, a higher prevalence (89 %) than reported in this study was found by [START_REF] Khoo | Coxiella detection in ticks from wildlife and livestock in Malaysia[END_REF]. According to the study carried out by [START_REF] Selmi | Molecular detection and genetic characterization of the potentially pathogenic Coxiella burnetii and the endosymbiotic Candidatus Midichloria mitochondrii in ticks infesting camels (Camelus dromedarius) from Tunisia[END_REF] the desertic areas reduce the ticks activity to the minimum, especially during summer, regarding the high average of temperature as well as the scarcity of animal species [START_REF] Selmi | Molecular detection and genetic characterization of the potentially pathogenic Coxiella burnetii and the endosymbiotic Candidatus Midichloria mitochondrii in ticks infesting camels (Camelus dromedarius) from Tunisia[END_REF].

However, the prevalence of Coxiella-like bacteria reported here or in other studies, where ticks were collected on host animals, are much higher than in studies reported in ticks collected by flagging over vegetation. Environmental H. longicornis ticks, collected in the Chungbuk province (middle of the Korean), showed a Coxiella-like prevalence of 2 % (2 /100) of H. longicornis [START_REF] Lee | Identification of the Coxiella sp . Detected from Haemaphysalis longicornis ticks in Korea[END_REF]; in Queensland, Australia, the prevalence reached 6 % (3/45) [START_REF] Seo | Molecular detection and genotyping of Coxiella-like endosymbionts in ticks that infest horses in south Korea[END_REF], 0.9 % (1 of 109) in Haemaphysalis punctata ticks from Spain [START_REF] Barandika | Prevalence of tick-borne zoonotic bacteria in questing adult ticks from northern Spain[END_REF]. Thus, Coxiella-like infection in ticks might be originated from host animals since its prevalence in environmental ticks is much lower than that described in ticks collected on animals.

The gender of the ticks does not appear to be a factor of genetic variability of the Coxiellalike since sequences belonging to the same clade were found in both male and female ticks.

According to the 16S rRNA phylogenetic analysis, three different clades of Coxiella-like bacteria occur in ticks from Algerian cattle. The dominant one, regrouping 11 of the 13 sequences identified seems to be genetically homogenous with few differences among each other (based on 16S rRNA gene). However, Coxiella-like bacteria from this genetic group were found in our study in different tick species (R. bursa, R. sanguineus SL-A, H. marginatum, H. excavatum, D. marginatus, H. sulcata and I. ricinus) and from different regions. This goes against the previous concept that each genetic variant of Coxiella-like bacteria is associated with specific tick species. The second 16S-based genetic group was identified in a female of R.

sanguineus SL-A (it is close to Coxiella endosymbiont of R. sanguineus SL-A from Brazil [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF] and Coxiella-like endosymbiont of H. excavatum from Anatolia, Turkey [START_REF] Brinkmann | A crosssectional screening by next-generation sequencing reveals Rickettsia , Theileria and Hemolivia species in ticks from Anatolia[END_REF]. The third genetic clade clustered with Coxiella endosymbiont of Rhipicephalus from Italy [START_REF] Duron | The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii[END_REF] (Fig. 3). Actually, the studies proofed the vertical transmission of CLE nevertheless; they do not deny the possible horizontal transmission, between ticks' species via infested cattle (the common point between them). For that, we hypothesized that the genetic diversities of the CLE don't only depend on the tick's species but also on the vertebrates' hosts.

Only four sequences were obtained for GroEL gene. This could be due to the primer's specificity and / or to the use of non-nested PCR protocol to detect Coxiella-like DNA which probably decreased, the PCR sensitivity. However, in our study, CLE DNA were successfully sequenced but some of sequences were impossibly assembled with the ChromasPro software due to their poor quality. We avoided the inclusion of these poor sequences in the analysis.

Conclusion

In our study, we report for the first time in Algeria that Coxiella-like 16S rRNA gene sequences from different tick species grouped into three clades. Beside the tick species specificities, the genetic diversity of CLE seems to be related to the vertebrate animal hosts, which support the possibility of horizontal transmission between ticks via the vertebrate animals.

CP0249 61 Fig. 1 .

 611 Fig. 1. Climate diagrams for the three Algerian provinces concerned by the tick collection, showing the long-term mean temperature of the coldest and the warmest month (Rubel, 2020, pers. communication).

Fig. 2 .

 2 Fig. 2. A map of northern Algeria showing different provinces from which the sample collections were carried out, with percentage of each tick species found and identified.(https://www.google.com/maps/@36.2736941,4.221111,230025m/data=!3m1!1e3?hl=en).

Fig. 3 .

 3 Fig. 3. Maximum-likelihood phylogenetic analysis of the Coxiella-like based on 16S rRNA gene sequences. Sequences characterized in this study are given in bold and indicatedwith the identification (number) of DNA sample, host tick species, its gender and the geographical origin, respectively. Legionella pneumophila subsp. pneumophila str. Philadelphia was included as the outgroup. Sequences were aligned using CLUSTALW, and phylogenetic inferences was conducted in MEGA6.06 using the maximum likelihood method based on the

  The best BLAST hit oscillated between 98.38 % and 99.63 %. Of the 4 sequences, two (samples 111 and 118) exhibited 98.38 and 98.63 % of identity with C. burnetii str. Schperling (CP014563). The other samples exhibited 98.49 and 98.92 % of identity with Candidatus Coxiella mudrowiae isolate CRS-CAT (CP024961) (Table 2).

	The phylogenetic analysis, based on the 16S rRNA gene, showed that Coxiella bacteria
	sequences from Rhipicephalus, Dermacentor, Ixodes and Hyalomma species (11 of 13
	successfully sequenced) are grouped into an independent and distinct clad. While the sequences
	from a female R. sanguineus SL-A from Blida remained distinct to others and share the same
	common ancestor with Coxiella endosymbiont of R. sanguineus SL-A isolate Rhsa 2 from

Brazil (KP994844). In addition, the 16S rRNA sequence of Coxiella-like of a male H. sulcata from Blida in this study clustered with the Coxiella endosymbiont of Rhipicephalus from Italy (KP994831) (Fig.

3

).

Phylogenetic analysis based on

GroEL partial gene of Coxiella-like bacteria was carried out. Two sequences gave similarity with C. burnetii by BLAST. They are grouped with C. burnetii Nine Mile strain and other C. burnetii strains. However, other sequences of Coxiellalike endosymbionts identified in R. sanguineus SL-A and H. excavatum were grouped with endosymbionts of R. sanguineus SL-A isolate Rhsa1 chaperonin (GroEL ) gene and share the same common ancestor (Fig.

  Haemaphysalis bispinosa: 18/18 adult females, among them five engorged, 2/2 unfed adult males and 1/1 nymph). In South Korea,[START_REF] Seo | Molecular detection and genotyping of Coxiella-like endosymbionts in ticks that infest horses in south Korea[END_REF] reported the prevalence of 52.4 % (121/231) of adult Haemaphysalis longicornis. These different data suggest that the Coxiella-like prevalence in ticks may depend, not only on the tick's species,

						reported a prevalence of 89%
	(49/55) of tested ticks (Haemaphysalis hystricis: 6/6 unfed adult females, 13/13 unfed adult
	males; Dermacentor	steini:	0/1	unfed	adult	females,	2/6	unfed	adult
	males; Dermacentor compactus: 1/2 unfed adult males; Amblyomma sp.: 5/5 engorged adult
	females, 1/1 unfed adult male;

  Blida and Batna successively. Whenever we were able, in 3 ticks, to amplify both genes of Coxiella-like bacteria (16S rRNA gene and GroEL ), we may suggest that GroEL sequences from ticks 11 and 118 (Fig.4) represent the same first dominant 16S clade and the sequences from ticks 110 and 146 are the sequences corresponding to the second 16S clade.Unfortunately, we were not able to amplify the GroEL gene from the only H. sulcata tick where the third 16S-based clade of Coxiella-like bacteria was identified.

Two sequences from female R. bursa ticks were almost identical to C. burnetii str.

Schperling detected on R. sanguineus SL-A female tick from Blida province. Two other sequences from Hyalomma ticks showed high similarity (>98 %) with Candidatus Coxiella mudrowiae isolate CRS-CAT detected on R. sanguineus SL-A and H. excavatum ticks from

Table 2 .

 2 BLAST analysis of specimens sequenced with 16S rRNA and GroEL partial 512 genes and selected to create the phylogenetic trees.

	513
								Best BLAST
	Primers	Localities	No of	DNA	Gender	of	Ticks species		The	Accession
			sequenced	samples	tick			hit	Percent	Numbers
			/ tested	ID					identity
	16S	Medea	1/3	5	Female		R. bursa	Coxiellaceae bacterium	99.39 %	KM079623
								CR07
		Blida	12 /16	34	Female		R. bursa	Coxiellaceae bacterium	99.60 %	KM0796
								CR07		23
				69	Male		H. sulcata	Uncultured Coxiella sp.	98.64 %	MK1640
								clone 195C		20
				74	Male		R. bursa	Uncultured Coxiella sp.	98.64 %	MK1640
								clone 195C		20
				75	Male		D. marginatus	Coxiellaceae	98.31 %	KM0796
								bacteriumCR07		23
				90	Male		R. bursa	Coxiellaceae bacterium	99.37 %	KM0796
								CR07		23
				106	Female		H. marginatum	Coxiellaceae bacterium	99.80 %	KM0796
								CR07		23
				110	Male		R. sanguineus SL-A	Coxiellaceae bacterium	100 %	KM0796
								CR07		23
				111	Female		R. bursa	Coxiellaceae bacterium	100 %	KM0796
								AL09		26
				113	Female		R. bursa	Coxiellaceae bacterium	100 %	KM0796
								CR07		23
				118	Male		R. bursa	Coxiellaceae bacterium	100 %	KM0796
								CR07

Acknowledgments

The authors would like to thank all the veterinarians who participated in the collection of the samples.

Funding

The project leading to this publication, received funding from the Excellence Initiative of Aix-Marseille University -A*MIDEX, a French scheme managed by the French National Research Agency under the "Investissements d'Avenir" program with reference number ANR-10-IAHU-03 and the Fondation Méditerranée Infection (www.mediterranee-infection.com).

This work was also supported by the framework of the CNEPRU university research project led by the university of Annaba Algeria, entitled biodiversity and parasitism of certain micromammals and migratory birds in wetlands, project code: D01N01UN230120150016.

Conflict of interest

The authors declare no conflicts of interest.

Ethical considerations

The collection of samples from cows was carried out in accordance with the Algerian legislation. Permission was obtained from the Ministry of Higher Education and Scientific Research of Algeria, in collaboration with the University Yahya Fares of Medea, and the National Veterinary High School, Algiers, Algeria.

Supplementary data

No.

Tm: Annealing temperature.