Pathogen Detection in Ornithodoros sonrai Ticks and Invasive House Mice Mus musculus domesticus in Senegal

Ornithodoros sonrai (O. sonrai) ticks are the only known vectors of Borrelia crocidurae, an agent of tick-borne relapsing fever (TBRF) borreliosis. Rodents serve as principal natural reservoirs for Borrelia. Our research objective was to detect TBRF Borrelia and other zoonotic bacterial infections in ticks and in house mice Mus musculus domesticus, an invasive species currently expanding in rural northern Senegal. Real-time and conventional PCR were utilized for detecting Borrelia and other bacterial taxa. The analyses were performed on 253 specimens of O. sonrai and 150 samples of brain and spleen tissue from rodents. Borrelia crocidurae was found in one O. sonrai tick and 18 Mus musculus domesticus samples, with prevalences of 0.39 percent and 12 percent, respectively, as well as Ehrlichia sp. in one Mus musculus domesticus. Further, we were able to detect the presence of a potentially infectious novel species belonging to the Anaplasmataceae family for the first time in O. sonrai ticks. More attention should be paid to the house mouse and O. sonrai ticks, as they can be potential hosts for novel species of pathogenic bacteria in humans.


Introduction
Ticks are strict blood-sucking arthropods divided into two families, Ixodidae (about 700 hard tick species) and Argasidae (around 200 soft tick species). The latter comprises the sub-families Argasinae and Ornithodorinae, which include the genus Ornithodoros [1,2]. Ticks can have a negative impact on an animal's health. They represent the second largest group for the transmission of infectious pathogens to humans after mosquitoes [3]. Tickborne relapsing fever (TBRF) borreliosis is caused by various species of spirochetes of the genus Borrelia, of which Borrelia crocidurae (Léger, 1917), Borrelia duttonii (Novy and Knapp, 1906) and Borrelia hispanica (de Buen, 1926) occur in Africa. These spirochetes are transmitted by soft ticks of the genus Ornithodoros [4][5][6]. As the name implies, TBRF is characterized by recurrent fever and associated symptoms, and in extreme cases the infection can spread to the neurological and cardiovascular systems [6]. In West Africa, TBRF is endemic in the Sahara, Sahel and Sudano-Sahelian regions [5]. In West and North Africa, small mammals are considered to be the principal reservoirs of Borrelia, particularly of B. crocidurae [7], which has been detected in several rodent species [5,7,8]. Borrelia crocidurae is transmitted via a bite by the tick Ornithodoros sonrai (Sautet and Witkowski, 1943 [5,7]) that lives inside small rodent burrows [9].
The purpose of this study was to gain a better understanding of the risk associated with the transmission of TBRF and other zoonotic bacteria carried by invasive small rodents in the rural area of Dodel, northern Senegal, and more particularly the domestic mouse Mus musculus domesticus (Schwarz and Scharz, 1943) [12]. We also sought to identify an expanded repertoire of infectious agents in order to increase the awareness of pathogens transmitted by O. sonrai soft ticks, hosted by small rodents, which may have a negative impact on human and animal health.

Study Area
The study was conducted in the rural commune of Dodel (16 • 29 16.7 N/14 • 25 19.0 W), located 531 km northeast of Dakar, in the Saint-Louis region ( Figure 1). In this area, the vegetation and climate are typically Sahelian, with a short wet season from July to mid-October. The survey included 60 housing plots visited for rodents and 34 for Ornithodoros ticks in 2018, as well as 77 and 10 others, respectively, in 2021 in Dodel's central area. These houses accommodate 489 people, the majority of them belonging to the Peulh "Pulaar" ethnic group. The population is more involved in trade than in agriculture and livestock.

Sampling
Rodents were captured alive using associated sets of locally-made wire-mesh and Sherman (H.B. Sherman Traps, Inc., Tallahassee, FL, USA) folding box traps placed within dwellings (bedrooms, hallways, kitchens, etc.) and shops. Trapping campaigns

Sampling
Rodents were captured alive using associated sets of locally-made wire-mesh and Sherman (H.B. Sherman Traps, Inc., Tallahassee, FL, USA) folding box traps placed within dwellings (bedrooms, hallways, kitchens, etc.) and shops. Trapping campaigns within private dwellings were conducted with prior explicit oral agreements from the relevant local authority (head of village and mayor) and individual householders after providing a comprehensive presentation of the project and its objectives. The traps were baited daily with groundnut paste and left for one to three days [7,9]. They were checked every morning and afternoon (while re-baiting) for night and day captures, respectively. All specimens caught were identified according to morphological criteria as previously reported [13]. Rodents were euthanized by performing cervical dislocation and handled in accordance with the relevant requirements of Senegalese legislation and the live animal capture guidelines of the American Society of Mammalogists (Animal Care and Use Committee 2011). Body measurements were taken before autopsy and reproductive status was noted. Brain and spleen tissue as well as digestive tracts were preserved in 95% ethanol for further analyses. All protocols used here were conducted following official regulations (Centre de Biologie pour la Gestion des Populations (CBGP)): Agrément pour l'utilisation d'animaux à des fins scientifiques D-34-169-003) of the relevant institutional committee (Regional Head of the Veterinary Service, Hérault, France). All transfer and conservation procedures were performed in accordance with current Senegalese and French legislation.
Ornithodoros ticks were collected from inside rodent burrows and crevices identified in domestic and peri-domestic environments using a modified portable petrol-powered aspirator [5]. They were kept in cryotubes containing 95% ethanol for the identification of their morphological features and further molecular analyses. A total of 104 rodent burrows were examined in dwellings and peri-domestic areas. The rodent burrow infestation rate was calculated as the ratio of the number of burrows harboring ticks to the total number of burrows sampled.

Ornithodoros Tick Identification and DNA Extraction
The morphological identification of Ornithodoros ticks was carried out based on a tick identification guide [1]. Observations were carried out using an optical microscope, (Zeiss Axio Zoom V16 (Zeiss, Marly-le-Roi, France)), and a scanning electron microscope (SEM) (Hitachi High-Technologies, Tokyo, Japan).
DNA extraction was performed on a small portion of spleen and brain tissue separately for each rodent, as well as on ticks, using an EZ1 DNA tissue extraction kit (Qiagen, Hilden, Germany), and following a previously described protocol [9,14,15]. All Ornithodoros ticks were subjected to standard PCR and sequencing to confirm morphological identification using the 16S tick gene [14].

Screening for Bacteria
Bacteria screening was performed on rodent brain and spleen tissue as well as on Ornithodoros ticks using a real-time PCR (qPCR) CFX96 system (Bio-Rad, Marnes-la-Coquette, France). Bacterial detection was applied to each sample to detect the presence of bacteria such as Anaplasma, Borrelia, Bartonella, C. burnetii and Rickettsia using specific primers and probes [16]. Positive and negative controls were used in each molecular reaction as previously described [16]. Samples with a cycle threshold of (Ct) < 36 were considered positive [16,17]. The DNA of samples positive in qPCR was amplified using standard PCR, then sequenced to identify the bacterial species [16,18]. The genes used to target the presence of Anaplasmataceae and Borrelia spp. in rodents and/or O. sonrai ticks were Anaplasmataceae 23S rRNA [16] and Borrelia 16S rRNA [19]. Only the samples from rodents captured in 2018 were tested for Anaplasmataceae. Samples positive for the Anaplasmataceae 23S rRNA gene were tested with an additional PCR specific to the genus Ehrlichia groEL [20] and the Anaplasmataceae 16S rRNA gene [21], and those positive for Borrelia spp. based on 16S_Bor rRNA were subjected to qPCR for the glpQ gene, specific to B. crocidurae, as previously described [10]. Once the PCR products were amplified, electrophoretic migration was performed for each PCR reaction in a 1.5% agarose gel stained with SYBR Safe TM (Thermo Fisher Scientific, Illkirch, France) and visualized using the ultraviolet imager ChemiDoc MP (Bio-Rad, Marnes-la-Coquette, France). The PCR products were purified and sequenced using a Big Dye Terminator (Thermo Fisher Scientific, Illkirch, France) and genetic analyzer ABI PRISM 3130 (Applied Biosystems, Courtaboeuf, France) [16] with ChromasPro software, version 1.34 (Technelysium Pty, Ltd., Tewantin, QL, Australia). The sequences obtained were analyzed and assembled for comparison with sequences from the GenBank database using the BLAST tool, as described previously [9,16]. The phylogenetic analyses were established using MEGA software, version 7.0.21, with 100 bootstrap replications [22]. The accession numbers assigned by GenBank to all our sequences are listed as follows: for the Anaplasmataceae 23S rRNA gene (Anaplasma sp. (MW790939) and Ehrlichia sp. (MW790940)); for the Anaplasmataceae 16S rRNA gene (Anaplasma sp. (MW790941) and Ehrlichia sp. (MW790942)) and finally for the gene Ehrlichia groEL (Ehrlichia sp. (MW791746)). Morphologically, all 253 ticks collected belonged to the species O. sonrai. A random selection of 18 O. sonrai tick specimens was performed for molecular confirmation. Using the tick 16S gene, we were able to amplify the DNA of 33.33% (6/18) of these ticks. These six specimens were successfully sequenced. The blast results confirmed the identity of O. sonrai, with identification percentages ranging from 98.39% to 99.75% (Table 1). Of 253 tick DNA extracts tested using qPCR, one was found positive for B. crocidurae infection, leading to a prevalence of 0.39%. This single tick found infected was collected in a rodent burrow examined inside a store where food goods are sold. The results of the qPCR and the standard PCR of Anaplasma spp. detected in O. sonrai are detailed in Table 2. All ticks studied were negative for Bartonella spp., C. burnetii and Rickettsia spp.

Detection of B. crocidurae Infection in Rodents
Among the 150 DNA samples from rodent brain and spleen tissues extracted separately and tested with qPCR, 18 Mus musculus domesticus were found to be infected with Borrelia spp., which were later confirmed to be B. crocidurae using qPCR for the B. crocidurae-specific glpQ gene, with a prevalence of 12% (18/150) and a mean Ct of 28.81 (Table 3). Then, we were able to amplify 69% (9/13) of the samples identified as positive for B. crocidurae (CP003426.1/JX292897.1) with standard PCR and sequencing ( Table 3). The other 31% (4/13) of samples found positive for Borrelia spp. according to the qPCR results could not be amplified using standard PCR, which may be due to PCR inhibitors [23]. All tested individuals of A. niloticus were negative for Borrelia spp.

Detection of Anaplasma spp. in Rodents
In Mus musculus domesticus, the qPCR results revealed 10.16% (6/59) and 6.78% (4/59) positive samples for Anaplasma spp. in the spleen and brain tissue of rodents, respectively. Standard PCR and sequencing were performed for the positive samples obtained from the qPCR. Blast showed that 10% (1/10) were identified as Ehrlichia sp. (Table 4). No rodent was found to be positive for Bartonella spp., C. burnetii or Rickettsia spp.

Discussion
Our study confirms the success of Mus musculus domesticus as an invasive species in rural areas of northern Senegal [12]. In November 2016, this species represented 85% of the small rodents captured in Dodel [24], while this proportion was approximately 98% in 2018 and 2021 (this study). The difference in relative abundance observed between the two years of sampling may be indicative of an increase in the population size of the species in this locality, a hypothesis that will have to be verified by performing further sampling. This expansion of house mice in rural areas of inner Senegal at the expense of native species has been shown to be associated with gastrointestinal helminth patterns, some of which likely favor this invasive success [24].
Using molecular tools, the present results also reveal a respective prevalence of B. crocidurae infection in one O. sonrai tick and 18 Mus musculus domesticus of 0.39% (1/253) and 12% (18/150), associated with an infestation rate of rodent burrows by ticks inside dwellings of 22.1% (23/104). Interestingly, the only O. sonrai tick found to be infected was from a burrow examined inside a shopkeeper's store that hosted this single individual. The rate of O. sonrai ticks found to be infected with B. crocidurae was quite low compared to previous epidemiological studies conducted further south in western Senegal (the localities of Niakhar and Dielmo-Ndiop, especially) [5,7]. Although the abundance of O. sonrai ticks in some Dodel dwellings was high, the average value of 22.1% of burrows occupied by ticks appears below those recorded elsewhere in Senegal, with infestation percentages of 32%, 37% and 41% in the three regions of the country [5].
In small mammals, the prevalence of Borrelia infection ranges from 1.7% to 27.8% in West Africa [7]. In regions known to be endemic to TBRF in Senegal, such as Dielmo, Dakar, Thies and Richard-Toll, the prevalence of Borrelia infection was in the range of 13.8%-50% in two species of small mammals (A. niloticus and Mus musculus domesticus) [7,[25][26][27]. The house mouse, acting as a natural reservoir of Borrelia, is a strictly commensal species in Senegal [13], and its abundant presence in small rodent communities, which maintains infection rates, makes it a risk factor for TBRF transmission [7]. Our survey reveals that B. crocidurae infection was more prevalent in house mice than in O. sonrai ticks. Indeed, the rate of B. crocidurae infection in O. sonrai ticks was particularly low. One may hypothesize that the contact between reservoir hosts and vector ticks may be rare, as O. sonrai ticks are essentially endophilic, while house mice often hide in closets, stacks of items in storerooms or other hiding places rather than in burrows.

Discussion
Our study confirms the success of Mus musculus domesticus as an invasive species in rural areas of northern Senegal [12]. In November 2016, this species represented 85% of the small rodents captured in Dodel [24], while this proportion was approximately 98% in 2018 and 2021 (this study). The difference in relative abundance observed between the two years of sampling may be indicative of an increase in the population size of the species in this locality, a hypothesis that will have to be verified by performing further sampling. This expansion of house mice in rural areas of inner Senegal at the expense of native species has been shown to be associated with gastrointestinal helminth patterns, some of which likely favor this invasive success [24].
Using molecular tools, the present results also reveal a respective prevalence of B. crocidurae infection in one O. sonrai tick and 18 Mus musculus domesticus of 0.39% (1/253) and 12% (18/150), associated with an infestation rate of rodent burrows by ticks inside dwellings of 22.1% (23/104). Interestingly, the only O. sonrai tick found to be infected was from a burrow examined inside a shopkeeper's store that hosted this single individual. The rate of O. sonrai ticks found to be infected with B. crocidurae was quite low compared to previous epidemiological studies conducted further south in western Senegal (the localities of Niakhar and Dielmo-Ndiop, especially) [5,7]. Although the abundance of O. sonrai ticks in some Dodel dwellings was high, the average value of 22.1% of burrows occupied by ticks appears below those recorded elsewhere in Senegal, with infestation percentages of 32%, 37% and 41% in the three regions of the country [5].
In small mammals, the prevalence of Borrelia infection ranges from 1.7% to 27.8% in West Africa [7]. In regions known to be endemic to TBRF in Senegal, such as Dielmo, Dakar, Thies and Richard-Toll, the prevalence of Borrelia infection was in the range of 13.8%-50% in two species of small mammals (A. niloticus and Mus musculus domesticus) [7,[25][26][27]. The house mouse, acting as a natural reservoir of Borrelia, is a strictly commensal species in Senegal [13], and its abundant presence in small rodent communities, which maintains infection rates, makes it a risk factor for TBRF transmission [7]. Our survey reveals that B. crocidurae infection was more prevalent in house mice than in O. sonrai ticks. Indeed, the rate of B. crocidurae infection in O. sonrai ticks was particularly low. One may hypothesize that the contact between reservoir hosts and vector ticks may be rare, as O. sonrai ticks are essentially endophilic, while house mice often hide in closets, stacks of items in storerooms or other hiding places rather than in burrows.
These organisms cause anaplasmoses that affect human and animal health and are often transmissible by ticks [2,16,30]. In this work, we also found a potential new species of Ehrlichia in brain and spleen tissues of house mice from northern Senegal (Table 4). This result corroborates that of Dahmana et al. [9], who detected, among several other pathogens (including B. crocidurae), a potential new Candidatus "Ehrlichia senegalensis" in splenic tissue of rodents from the same area.
In Senegal, previous studies have reported the presence of Anaplasmataceae, with a high prevalence in small rodents [31,32]. In our study, we were able to amplify two potential new species, namely Anaplasma sp. and Ehrlichia sp., in O. sonrai ticks. In the phylogenetic trees, our potential new species of Anaplasma forms a separate cluster from other Anaplasma spp. (Figures 2 and 3), which means that it does not conform to any previously recognized species, thus potentially representing a new species. Concerning our amplicon of Ehrlichia sp., based on the two genes Anaplasmataceae rRNA 23S and Anaplasmataceae rRNA 16S, our sequence was 100% identical with Candidatus Ehrlichia sp. and uncultured Ehrlichia sp., respectively. The latter represents a potential new species that has not yet been described (Figures 2 and 3) [9]. With the Ehrlichia groEL gene, our amplicon forms a distinct cluster on the tree that could not be attributed to already recognized species (Figure 3). The detection of Anaplasmataceae in O. sonrai could be the consequence of a bacterial engorgement in ticks following a mixed infestation. This hypothesis has already been proposed for other tick species [33,34]. The vector competence of O. sonrai to transmit Anaplasmataceae cannot yet be confirmed, but there is a risk that it could be potentially a vector of this bacterium. Further studies and experimentation are needed to better understand the epidemiological relationship between Anaplasmataceae and these soft ticks.

Conclusions
This study contributes to the identification of an expanded repertoire of zoonotic bacteria hosted in Mus musculus domesticus house mice and O. sonrai ticks in northern Senegal. Ornithodoros sonrai ticks may be a potential vector of Anaplasmataceae. Further indepth investigation and experimentation should be conducted to understand the potential epidemiological role of these ticks and their impact on human and animal health.

Funding:
The field surveys for this study were carried out with funding from the UMR CBGP (Centre de Biologie pour la Gestion des Populations) and the ObsMiCE (West African Observatory of Small Mammals Indicators of Environmental Change) network from UMRs LPED and CBGP. The analyses using PCR standard and qPCR kits were supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the French National Research Agency under the "Investissements d'avenir" program, reference ANR-10-IAHU-03, the Provence-Alpes-Côte-d'Azur region and European regional development funding of platforms research and innovation mutualized Mediterranean Infection (FERDF PRIMMI).

Data Availability Statement:
Summary data on small mammals can be publicly accessed via http: //BPM-CBGP.science (accessed on 11 October 2022).