Detection of relapsing fever Borrelia spp., Bartonella spp. and Anaplasmataceae bacteria in argasid ticks in Algeria

Background Argasid ticks (soft ticks) are blood-feeding arthropods that can parasitize rodents, birds, humans, livestock and companion animals. Ticks of the Ornithodoros genus are known to be vectors of relapsing fever borreliosis in humans. In Algeria, little is known about relapsing fever borreliosis and other bacterial pathogens transmitted by argasid ticks. Methodology/Principal findings Between May 2013 and October 2015, we investigated the presence of soft ticks in 20 rodent burrows, 10 yellow-legged gull (Larus michahellis) nests and animal shelters in six locations in two different bioclimatic zones in Algeria. Six species of argasid ticks were identified morphologically and through 16S rRNA gene sequencing. The presence and prevalence of Borrelia spp., Bartonella spp., Rickettsia spp. and Anaplasmataceae was assessed by qPCR template assays in each specimen. All qPCR-positive samples were confirmed by standard PCR, followed by sequencing the amplified fragments. Two Borrelia species were identified: Borrelia hispanica in Ornithodoros occidentalis in Mostaganem, and Borrelia cf. turicatae in Carios capensis in Algiers. One new Bartonella genotype and one new Anaplasmataceae genotype were also identified in Argas persicus. Conclusions The present study highlights the presence of relapsing fever borreliosis agents, although this disease is rarely diagnosed in Algeria. Other bacteria of unknown pathogenicity detected in argasid ticks which may bite humans deserve further investigation.


Introduction
Ticks are obligatory hematophagous ectoparasites that can be vectors of protozoa, viruses and bacteria during their feeding process on animal hosts. They are currently considered to be second only to mosquitoes as vectors of human infectious disease around the world [1]. Two families of ticks are of medical significance: Ixodidae (hard ticks) and Argasidae (soft ticks). Hard ticks are the main ticks acting as vectors of human disease, but soft ticks are also known to transmit agents of human infectious diseases which are often neglected [2]. Argasid ticks comprise four genera and about 185 species, including three genera represented by a large range of species: Carios (87 species), Argas (57 species) and Ornithodoros (36 species). The fourth genus (Otobius) is represented by three species. This classification and the taxonomy of Argasids may evolve with the use of molecular tools [3]. Argasid ticks of the genus Ornithodoros include vectors of relapsing fever caused by Borrelia spp. in humans [4]. All these tick vectors are, for the most part, geographically restricted and are considered to be specific vectors of a given Borrelia spp. As Borrelia spp. may persist for many years in their long-lived vectors, Ornithodoros spp. are considered as both vectors and de facto reservoirs [2]. Vertebrate reservoirs of tick-borne relapsing fever (TBRF), Borrelia spp. include a variety of mammals, mainly rodents and insectivores, which inhabit burrows, dens and caves [5]. In Africa, TBRF Borrelia spp. include neglected vector-borne pathogens responsible for various febrile presentations and are most commonly suspected in malaria-like symptoms [6]. Tick-borne relapsing fever has been recognized as major cause of disease and death in several regions of Africa [7]. Currently, two agents of tick-borne relapsing fever have been detected in North Africa, namely Borrelia crocidurae and Borrelia hispanica [8]. An uncultured bacterium, "Borrelia merionesi," has also been detected in Ornithodoros ticks in Morocco [9], and "Candidatus Borrelia algerica" has been reported in febrile patients in Algeria [10]. In Algeria, since the first human case of TBRF reported by Sergent in 1908 [11], the disease has been largely neglected, and recent epidemiological data are lacking. The local transmission of TBRF is not known, and cases are not diagnosed. In addition to "Candidatus Borrelia algerica" which has been detected in Oran, Borrelia crocidurae has been detected in O. sonrai (2.5% prevalence) [12]. No other bacteria are known to be associated with soft ticks in this area.
In this paper, we report a series of entomological investigations that we conducted between 2012 and 2015. We aimed to describe the distribution of soft ticks in Algeria and the prevalence of associated Borrelia, Bartonella, Rickettsia and Anaplasma.

Ethical considerations
The study protocol was approved by the Steering Committee of the Algerian Ministry of Health (Direction Générale de la Prevention). Tick collection excluded national parks and protected areas and did not involve endangered or protected species (CITES, IUCN and national guidelines). All tick collections which took place inside homes and on private land were conducted after receiving permission from the owner.

Tick sampling
This study was carried out between May 2012 and October 2015 in six locations in two different bioclimatic zones in Algeria. We conducted several sampling series in three northern coastal areas with a Mediterranean climate (Mostaganem, Algiers and El Tarf) and one in the highlands with a semi-arid climate (M'sila).
In Mostaganem, on the western coast of Algeria, three sites were investigated, including the port of Mostaganem (35˚53'39" N, 0˚05'25" E), Sidi Ali (36˚06' 00" N, 0˚25'00" E) and Achaacha (36˚14' 47" N, 0˚38'03" E). In Achaacha in March 2015Achaacha, we also specifically investigated rodent burrows within a farm. The owner of the farm had been hospitalized with an unexplained fever the previous year. Argasid ticks were also collected in Algiers from the central coastal area (the island of Agueli) (36˚44' 00" N, 3˚21'00" E), El Tarf (36˚42' 2" N, 81 8'50" E) and M'sila in the eastern highlands of Algeria (35˚35' 13" N, 4˚40'08" E). Ticks were sampled from a variety of natural and human-impacted habitats. In this study, we prospected rodent burrows and yellow-legged gull (Larus michahellis) nests, and inspected other animal shelters. We introduced a flexible tube into rodent burrows and aspirated the contents using a portable, petrol-powered aspirator [12]. Ticks from seabird nests were collected after the nesting period; the nests were recovered when the chicks had left their nests. The seabird nests were collected from between the rookeries on the island of Agueli (Algiers). The nests were then placed in individual bags to avoid parasite loss. Ticks were collected by sifting through the contents. Ticks were collected from slits inside animal shelters using entomological forceps. After collection, all biological materials were immediately stored in ethanol or liquid nitrogen (one tube per positive burrow/nest/animal shelter) and then forwarded to Marseille, France.

Identification of ticks and the molecular detection of bacteria
Identification and molecular detection. Ticks were identified by morphological criteria using standard taxonomic keys [13]. Each specimen was rinsed twice in distilled water for 10 minutes and then dried on sterile filter paper. Handling was performed in a laminar flow biosafety cabinet. Ticks were individually crushed in sterile Eppendorf tubes. Total DNA was extracted in a final volume of 200 μl from one half of each ectoparasite using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) by Qiagen-BioRobot EZ1 according to the manufacturer's instructions [14]. Genomic DNA was stored at -20˚C under sterile conditions. Once the DNA had been extracted, ticks were also identified by 16S rRNA gene sequencing, as previously described [12]. The DNA of each specimen was then used in qPCR template assays to detect Bartonella spp., Rickettsia spp., Anaplasmataceae bacteria and Borrelia spp. [15]. Negative controls were used in each qPCR and consisted of DNA extracted from uninfected ticks from our laboratory colony. Positive controls included DNA extracted from a dilution of cultured strains of Borrelia crocidurae (for the detection of Borrelia spp.), Bartonella henselae (for the detection of Bartonella spp.), Rickettsia montanensis (for the detection of Rickettsia spp.) and Ehrlichia canis (for the detection of Anaplasmataceae bacteria). Results were deemed positive if the Cycle threshold (Ct) value obtained by CFX96 was lower than 36. All samples identified as positive by qPCR (16S rDNA-based) for Borrelia spp. were confirmed by a standard PCR and sequencing for the fragments of the flagellin gene (flaB) [10]. For the detection of Bartonella spp., we targeted the 16S/23S RNA intergenic spacer (ITS) gene for qPCR [16] and confirmed the partial sequence of the citrate synthase gene (gltA) and the cell division protein gene (ftsZ) by sequencing. For the detection of Rickettsia spp., we targeted a partial sequence of the citrate synthase (gltA) (RKND03 system; Rickettsia genus-specific) for qPCR. Positive samples were tested by standard PCR targeting the gltA [17]. For detection of Anaplasmataceae bacteria, we targeted the 23S rRNA gene, as described [18] (Table 1). DNA sequencing reactions were performed on highly positive samples (Ct <28).

Phylogenetic analysis
Phylogenetic trees were drawn using the neighbor-joining method from an alignment of the different genes used in the experiments. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the ML phylogenetic analysis with the TOPALi 2.5 software (Biomathematics and Statistics Scotland, Edinburgh, UK) within the integrated ML application, using the K81uf + I + Г substitution model. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis from 100 replicates to generate a majority consensus tree.

Sample collection and tick identification
In this study, 20 rodent burrows, 10 yellow-legged gull (Larus michahellis) nests and three animal shelters were prospectively sampled. A total of 204 ticks were collected at six sites. The identification of ticks using entomological keys and molecular tools is reported in Table 1. Six distinct species belonging to three genera were identified, including Carios capensis, Ornithodoros rupestris, Ornithodoros occidentalis, Ornithodoros erraticus, Ornithodoros sonrai and Argas persicus (Fig 1).
Detection of Bartonella spp., Rickettsia spp. and Anaplasmataceae bacteria Detection of bacterial agents in argasid ticks in Algeria ticks tested positive for Bartonella spp. These four ticks tested positive by standard PCR using primers targeting the ftsZ gene, and two were positive by PCR using gltA gene primers. Using DNA sequencing, we identified four ftsZ and two gltA sequences of Bartonella spp. (Table 1). The closest sequence available in GenBank was that of Bartonella elizabethae (AF467760; 96% similarity) from the American Type Culture Collection, Manassas VA, USA (F9251T/ATCC. 49927). A phylogenetic tree based on concatenated ftsZ (292 bps) and gltA (200 bps) genes showed that our genotype belongs to a cluster of Bartonella spp. including Bartonella bovis, Bartonella capreoli, Bartonella schoenbuchensis and Bartonella chomelii (Fig 3). Furthermore, standard PCR revealed that 17 samples tested positive for Anaplasmataceae DNA (23S rRNA gene). We also identified six DNA sequences of Anaplasmataceae bacteria in six Argas persicus GenBank accession numbers are indicated at the beginning. Sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using the ML phylogenetic analysis with TOPALi 2.5 software (Biomathematics and Statistics Scotland, Edinburgh, UK) within the integrated ML application, using the K81uf + I + Г substitution model. Numbers at the nodes are percentages of bootstrap values obtained by repeating the analysis 100 times to generate a majority consensus tree (only those equal or higher than 80 were retained). The final set includes 492 base pairs. The scale bar represents a 20% nucleotide sequence divergence. Genbank accession numbers of ftsZ and gltA genes respectively are indicated at the beginning.  (Table 1). The closest sequence available in GenBank was the sequence of Anaplasma ovis (KM021411.1; 93% similarity), isolated from sheep in Senegal. Although one sample tested positive for Rickettsia DNA (the gltA gene), the quality of the sequence obtained was too poor to be interpreted.

Discussion
This investigation reports direct evidence of DNA from Borrelia spp., Bartonella spp., and an Anaplasmataceae bacterium in argasid ticks in Algeria.
In this study, we identified Borrelia DNA that, based on analysis of a portion of the flaB gene, is identical to B. turicatae. Few studies have been performed and little molecular data exists for B. turicatae, related species and isolates [4]. Borrelia turicatae is known as a New World agent of tick-borne relapsing fever, which is prevalent throughout the southern United States and Latin America, and maintained there in enzootic cycles by a soft tick, O. turicata [19]. This tick is present throughout Mexico and Central and South America, where it colonizes peridomestic settings and inhabits burrows, nests, caves, and cavities under rocky outcrops. O. turicata are also promiscuous feeders, and recognized hosts include prairie dogs, ground squirrels, snakes, cattle, pigs, and the gopher tortoise [19]. Borrelia turicatae is genetically closely related to B. parkeri, another New World agent of tick-borne relapsing fever. However, B. parkeri differs from both B. turicatae and B. hermsii, a causative agent of tickborne relapsing fever in the western United States, by the lack of circular plasmids in its genome [20].
Our results, reporting the infection of C. capensis collected from Algerian seabird nests with Borrelia turicatae, might be considered surprising, as they do not support the tick-spirochete species specificity that has long been highlighted by previous authors. C. capensis are typically nest-associated ticks with a strong specificity for seabirds. They are known to infest the nests of the yellow-legged gull (Larus michahellis) along the coasts of Algeria [21]. However, our results are supported by the analysis of a portion of the flaB gene only. Borrelial flagellin is encoded by the flaB gene, which is genus-specific and highly conservative among Borrelia spp. Interestingly, the sequences of the flagellin gene may be identical in Borrelia spp. although they have different tick hosts, different epidemiology, and profound genomic differences (i.e., B. recurrentis, the agent of louse-borne relapsing fever, and B. duttonii, the agent of East-African tick-borne relapsing fever) [6,22]. However, due to a lack of DNA, we were unable to analyze more genes of the Borrelia spp. in this study. This is why we reference the genotype identified in this work as originating from Borrelia cf. turicatae.
Moreover, several Borrelia sequences which are very closely related to Borrelia turicatae sequences were recently reported from bat ticks in the USA [23]. An almost identical bacterium was also once found in another seagull-associated soft tick, Carios sawaii in Japan [24], as well as in African penguins [25]. Hence, it would appear that several Borrelia spp. closely related to B. turicatae are associated with seabirds and their ticks [25]. Our results offer additional evidence of this association and the consequences deserve further investigation. Carios capensis may also pose a threat to humans visiting yellow-legged gull rookeries if visitors are exposed to ticks harboring infectious agents.
Borrelia hispanica is an agent of tick-borne relapsing fever in the Old World. It is known to be transmitted to humans by infected O. erraticus ticks in Spain, Portugal, and Morocco [26]. In the Iberian Peninsula, this tick is associated with swine and rodents and their surroundings, where ticks remain buried in soil or crevices. Recently, Borrelia hispanica was found in O. marocanus (5/43, 11.6%) and O. occidentalis (3/55, 5.5%) in Morocco, and O. kairouanensis (1/5, 20.0%) in Tunisia [5]. In this study, Borrelia hispanica was detected for the first time in Algeria in O. occidentalis (2/6, 33.3%) collected from a farm owned by a patient with a previously unexplained fever in Achaacha (Mostaganem). Recently, O. occidentalis has been reported to belong, alongside O. marocanus and O. kairouanensis, to a complex of three large species (female adult length 5.5-7.5 mm) distributed in typically Mediterranean areas of Morocco, Algeria, Tunisia and Spain and previously confused with O. erraticus [12]. Interestingly, it has been reported that in northwestern Morocco, 20.5% of patients presenting with an unexplained fever had tick-borne relapsing fever related to Borrelia hispanica [27]. However, further epidemiological studies are recommended to screen patients with unexplained fever in Algeria.
Human illnesses associated with Bartonella occur worldwide, and they encompass a broad clinical spectrum, including fever, skin lesions, endocarditis, lymphadenopathy, and abnormalities of the central nervous system, eye, liver and bone [28]. Bartonelloses are infectious diseases caused by bacteria from the genus Bartonella which infects erythrocytes and endothelial cells in humans [29]. Biting arthropod vectors transmit these pathogens and infect a wide range of wild and domestic mammals, including rodents, cats, dogs, and cattle. Throughout our investigation, we identified A. persicus Bartonella spp. in 2/50 (4%) fowl ticks by sequence analysis of ftsZ and gltA. The phylogenetic tree demonstrates that our genotype belongs to the cluster of the Bartonella spp. family, including Bartonella bovis transmitted by Haemaphysalis bispinosa [30], Bartonella capreoli transmitted by Ixodes ricinus [31], Bartonella schoenbuchensis transmitted by Melophagus ovinus, and Lipoptena cervi for Bartonella chomelii [32].
Argas persicus, currently considered native to Turanian-Central Asia, is a parasite of arboreal nesting birds, and has successfully adapted to coexist with domestic fowl. Probably as the result of human transport, it has spread worldwide, where it survives practically exclusively in association with domestic fowl [33]. Its bite is painful to humans, often with toxic after-effects, but such episodes in humans are rare [34]. The fowl tick has been morphologically described previously in the Mediterranean basin and in Algeria [13]. It may transmit avian encephalomyelitis, leucocytozoonosis [35], and West Nile virus [36]. A. persicus could harbor different types of bacteria, including those of the genus Salmonella, Aerobacter, Escherichia, Proteus, Staphylococcus, Flavobacterium, Bacillus, Pseudomonas, and Streptococcus [37]. Here, the association between Bartonella sp. and A. persicus is reported for the first time in Algeria. Since ticks have been collected from animals that, in some cases, could be bacteremic, the ticks may be vectors for some pathogens, but also only carriers in other cases. Consequently, we cannot consider the presence of bacteria in an arthropod as proof of vector competence.
Anaplasma spp. are globally-distributed tick-transmitted bacteria of importance to veterinary and public health. These pathogens cause anaplasmosis in domestic and wild animal species, as well as in humans. Rhipicephalus, Ixodes, Dermacentor and Amblyomma genera of hard ticks include vectors of Anaplasma spp. [38]. Here, we identified a new genotype of Anaplasmataceae bacteria in 6/50 Argas persicus. In the phylogenetic tree, Anaplasma amplified from ticks collected in this study were closely related to "Candidatus Anaplasma ivoirensis" (KT364326) detected in Amblyomma variegatum ticks in Cote d'Ivoire, Anaplasma ovis strain KMND (KM021411) detected in sheep in Senegal [39], and Anaplasma marginale (TCI219) detected from Rhipicephalus microplus in Cote d'Ivoire (Fig 4). The pathogenicity of the Anaplasma genotype detected in our study is still unknown, although the closely related Anaplasma marginale is a globally-distributed tick-borne disease agent with great economic importance in the world's cattle industry [40]. Interestingly, in vitro cultivation of A. marginale and A. phagocytophilum in argasid ticks cell lines has been achieved [41]. As discussed above, the detection of DNA in a tick does not mean that this tick is an efficient vector of the bacteria. There is a lack of information about Anaplasmataceae infection in argasid ticks, and our findings represent the first report of argasid tick infection with Anaplasma in Algeria. Unfortunately, due to the lack of DNA, we were unable to amplify and sequence other gene fragments in order to better characterize our Anaplasmataceae genotypes as belonging to new species. Phylogenetic inferences were obtained using MEGA 6. GenBank accession numbers are indicated at the beginning and the geographic origin of the species is indicated at the end. The 23S rRNA gene sequences were aligned using CLUSTALW, and phylogenetic inferences were obtained using the ML phylogenetic analysis with TOPALi 2.5 software (Biomathematics and Statistics Scotland, Edinburgh, UK) within the integrated ML application, using the K81uf + I + Г substitution model. https://doi.org/10.1371/journal.pntd.0006064.g004

Detection of bacterial agents in argasid ticks in Algeria
Finally, for the first time in Algeria, we detected Borrelia cf. turicatae in C. capensis, and Borrelia hispanica in O. occidentalis, in two coastal regions of Algeria. We also detected one genus of Bartonella spp. and one genus of Anaplasmataceae bacteria in A. persicus. We have expanded the understanding of the repertoire of argasid ticks with the first molecular evidence of A. persicus in Mostaganem and tick-borne bacteria present in these arthropods in Algeria. Our findings will help human and veterinary clinicians to enlarge the spectrum of pathogens to be considered in differential diagnosis. Further work is needed to map tick/borrelia distribution in relation to environmental and climatic characteristics.