The Surveillance of Borrelia Species in Camelus dromedarius and Associated Ticks: The First Detection of Borrelia miyamotoi in Egypt

Simple Summary Lyme borreliosis (LB) is a zoonotic disease caused by the spirochete Borrelia burgdorferi sensu lato (s.l.) transmitted to humans by the bite of infected Ixodes ticks. Borrelia miyamotoi is a spirochete that causes relapsing fever (RF) and is genetically related to Borrelia burgdorferi s.l. It is the only Borrelia species in the RF group that can be spread by the Ixodes tick. However, there have been no reports of B. miyamotoi in Egypt, and the data on LB in camels is scarce. Therefore, the current study is the first molecular study for the detection of Borrelia spp. and Borrelia miyamotoi in camels and associated ticks in Egypt. Furthermore, the identification of tick species that feed on camels demonstrates the value of cox1-based molecular approaches for identifying tick species. Here, we provide the first insight into the Borrelia miyamotoi and B. afzelii found in Egyptian camels and related ticks. Thus, it is essential to comprehend the role of the host in transmission and to constantly monitor the emergence of new diseases in Egypt. Abstract Tick-borne diseases (TBDs) are emerging and re-emerging infections that have a worldwide impact on human and animal health. Lyme borreliosis (LB) is a severe zoonotic disease caused by the spirochete Borrelia burgdorferi sensu lato (s.l.) transmitted to humans by the bite of infected Ixodes ticks. Borrelia miyamotoi is a spirochete that causes relapsing fever (RF) and is genetically related to Borrelia burgdorferi s.l. However, there have been no reports of B. miyamotoi in Egypt, and the data on LB in camels is scarce. Thus, the present study was conducted to screen and genetically identify Borrelia spp. and B. miyamotoi in Egyptian camels and associated ticks using polymerase chain reaction (PCR). Methods: A total of 133 blood samples and 1596 adult hard ticks were collected from Camelus dromedaries at Cairo and Giza slaughterhouses in Egypt. Tick species were identified by examining their morphology and sequencing the cytochrome C oxidase subunit 1 (cox1) gene. Borrelia spp. was detected using nested PCR on the IGS (16S-23S) gene, and positive samples were genotyped using 16S rRNA and glpQ spp. genes specific for Borrelia burgdorferi and Borrelia miyamotoi, respectively. The positive PCR products were sequenced and analyzed by phylogenetic tree. Results: Analysis of the cox1 gene sequence revealed that the adult ticks belonged to three genera; Hyalomma (H), Amblyomma (Am), and Rhipicephalus (R), as well as 12 species, including H. dromedarii, H. marginatum, H. excavatum, H. anatolicum, R. annulatus, R. pulchellus, Am. testudinarium, Am. hebraeum, Am. lipidium, Am. variegatum, Am. cohaerens and Am. gemma. Borrelia spp. was found in 8.3% (11/133) of the camel blood samples and 1.3% (21/1596) of the ticks, respectively. Sequencing of the IGS (16S-23S) gene found that B. afzelii, detected from H. dromedarii and H. marginatum, and B. crocidurae, which belongs to the RF group, was detected from one blood sample. B. burgdorferi and B. miyamotoi were discovered in the blood samples and tick species. Phylogenetic analysis of the glpQ gene showed that the B. miyamotoi in this study was of the Asian and European types. Conclusions: These results suggest that the camels can be infected by Lyme borrelia and other Borrelia bacteria species. This study also provides the first insight into the presence of Borrelia miyamotoi and B. afzelii DNA in camels and associated ticks in Egypt.


Background
The world population of Camelus dromedaries (one-hump dromedary camel) is estimated to be over 30 million, with Africa and the Middle East having the largest populations [1]. They are necessary for milk, meat, leather, transportation, and entertainment including tourism in Egypt. As a result, they play a critical role in the socio-economic development of many countries [2,3]. Because these camels are susceptible to several infectious illnesses, eating camel meat or having contact with them represents a significant source of zoonotic disease [1,4]. Tick infestations and TBDs are the most serious threat to camel health, causing global financial losses due to the geographic expansion of their tick vectors [5][6][7]. Amblyomma, Haemaphysalis, Hyalomma, and Rhipicephalus are the four genera of hard ticks that affect domestic animals in Egypt [8][9][10]. Moreover, tick species that severely infest dromedary camels in Egypt include H. dromedarii, H. excavatum, H. marginatum, and H. impeltatum [10][11][12]. Ticks carry a wide range of zoonotic pathogens, including Borrelia spp., which cause Lyme borreliosis (LB) and relapsing fever (RF) in humans [13]. Nearly 20 species within the Borrelia burgdorferi (sensu lato) complex were included, nine of which are known to cause animal and human LB (i.e., Borrelia afzelii, Borrelia bavariensis, Borrelia bissettii, B. burgdorferi (s.s.), Borrelia garinii, Borrelia kurtenbachii, Borrelia lusitaniae, Borrelia spielmanii and Borrelia valaisiana) [14]. Lyme borreliosis, which is spread naturally by Ixodes spp. ticks causes serious illness in humans, ranging from relatively benign skin lesions to severe cardiac, rheumatic, and neurologic signs [15][16][17]. Borrelia burgdorferi s.l. is widespread and is maintained in nature by various arthropod vectors, mammalian, birds, rats, reptiles, and many other wild species [18][19][20].
Borrelia miyamotoi is a spirochete that is genetically related to Borrelia burgdorferi s.l. It is the only Borrelia species in the RF group that can be spread by the Ixodes tick [21][22][23]. Borrelia miyamotoi infection commonly manifests as a febrile illness accompanied by fatigue, headache, chills, myalgia, arthralgia, and nausea, with potentially fatal complications such as meningoencephalitis [22,[24][25][26]. Ticks of all stages are possible vectors of B. miyamotoi because it has transstadial and transovarial transmission which is not the case with Lyme borrelia, indicating that the global distribution of B. miyamotoi-infected ticks may exceed that of B. burgdorferi-infected ticks [27,28]. B. miyamotoi has been divided into three groupings based on geographic areas and principal vector species: Asian (or Siberian) (transmitted by I. persulcatus and I. pavlovskyi), American (transmitted by I. scapularis and I. pacificus), and European (carried by I. ricinus) [29]; and a new fourth clade was recently found in I. ovatus ticks in Japan [30,31]. In Africa, RF is most common in the northern part of Africa and is caused by various Borrelia spp. such as B. hispanica, B. duttonii, and B. crocidurae [32]. However, a new species identified as B. miyamotoi has lately sparked renewed attention in this bacterial group [33]. These diseases are challenging to diagnose due to the nonspecific nature of the febrile illness, isolation difficulties, and cross-reactivity between serological techniques [12]. Therefore, it is critical to understand host transmission and to monitor for the emergence of new diseases [34]. Borrelia miyamotoi and other relapsing fever group members are distinguished by a glycerophosphodiester phosphodiesterase (glpQ) gene [22]. B. burgdorferi s.l. lacks the gene for glpQ; therefore, this gene is used to detect B. miyamotoi. In both B. burgdorferi s.l. and B. miyamotoi, an intergenic spacer (IGS) between the 16S and 23S genes is usually used to detect them [35,36].
There have been no reports of B. miyamotoi in Egypt, and information on LB in camels is limited. Thus, the present study was conducted to screen and genetically identify Borrelia spp. and B. miyamotoi in Egyptian camels and associated ticks using molecular methods. Furthermore, to investigate the status of hard tick species that infest Egyptian camels.

Sample Collection and Preparation
A total of 133 healthy one-humped camels (Camelus dromedarius) aged 3-5 years were investigated for tick infestation. Blood samples and hard ticks were collected from 50 and 83 healthy dromedary camels at Cairo and Giza slaughterhouses in Egypt, respectively.
The study was conducted from February 2021 to November 2021. Blood samples (5 mL) were collected in tubes coated with EDTA from the jugular blood vessels of the examined camels, then transferred to the laboratory in an icebox and maintained at −20 • C until DNA extraction. A total of 1596 adult ticks (600 from Cairo and 996 from Giza) were carefully gathered from the camels, then transferred alive to the zoonoses laboratory. Adult ticks were washed twice in distilled water, dried with paper tissues, and the classification of all collected ticks to genus level was by morphological characteristics using a stereomicroscope and taxonomic keys [37].

Extraction of DNA from Blood Samples and Ticks
Morphologically identified ticks (3-5 ticks/genus) were crushed into small pieces in a mortar with liquid nitrogen, then DNA was extracted from the ticks and blood samples (200 µL) using a Thermo Scientific GeneJET Genomic DNA Purification Kit (ThermoFisher, Darmstadt, Germany) according to the manufacturer's recommended protocol. Isolated material was stored at −20 • C until further molecular analysis.

Molecular Identification of Tick Species
Ticks were identified to species level by amplifying and sequencing an~820 bp fragment of the cytochrome oxidase c subunit I (cox1) gene. Amplification conditioning was performed according to [38]. Table 1 lists the primers used in this study.

Molecular Identification of the Borrelia Species
A nested PCR to detect Borrelia spp. in ticks and camel blood samples was carried out using outer and inner primers to amplify the 16S-23S rRNA intergenic spacer (16S-23S IGS) [40], and the amplification was performed according to [36].
Positive samples of Borrelia spp. were examined for detection of B. burgdorferi using a conventional PCR with a primer set of BbF and BbR for the 16S rRNA gene, according to [39]. Positive samples were further processed for sequencing.
An additional nested PCR test targeting the glpQ gene was performed on the positive samples to confirm the presence of B. miyamotoi and to detect genospecies. PCR amplicons were sequenced unidirectionally using primer Q3 to confirm the presence of B. miyamotoi in PCR-positive ticks and blood DNA samples [35,36].
The primers and amplification conditions for each reaction are displayed in Table 1. For amplification reactions cosmo Taq DNA Polymerase master mix (Willowfort, UK) was used. Each set of reactions included a positive control and the negative control consisted of nuclease-free water added to the PCR mix instead of the DNA sample. PCR products were visualized on 1.5% agarose gels.

Prevalence of the Borrelia Species in the Camel Blood Samples and Ticks
The camel blood samples and ticks were screened for the presence of Borrelia spp. using nested PCRs with the IGS gene and showed that the prevalence of Borrelia spp. in adult ticks was 1.3% (21/1596), whereas it was 8.3% (11/133) in the camel blood samples ( Table 3). Sequencing of the amplicons revealed the presence of three Borrelia spp. in the examined ticks, B. afzelii and B. burgdorferi (these two genospecies belonged to the B. burgdorferi s. l. group), and B. miyamotoi (belonging to the RF group). While B. burgdorferi, B. miyamotoi, and B. crocidurae were detected in blood samples (Table 3), Borrelia afzelii was not found in blood samples but found in two tick species, while B. crocidurae was detected in only one blood sample.

Phylogenetic analysis
Tick species were classified into three genera based on sequence analysis of the positive PCR products of the cox1 gene: Hyalomma, Amblyomma, and Rhipicephalus; this was validated using a sequence identity of 96 to 100% with tick species sequences in GenBank ( Figure 1). The accession numbers of cox1 gene sequences of identified ticks were deposited in GenBank, listed in Table 5.   The phylogenetic tree of Borrelia spp. based on the 16S-23S IGS gene was consistent with the sequence results, identifying four genospecies found in ticks and blood; B. burgdor-feri, B. afzelii, B. miyamotoi, and B. crocidurae. The B. miyamotoi found in Am. lepidum and Am. variegatum was identical to a German isolate (GenBank: MK945853.1, MK945806.1, MK945787.1, MK458687.1) and clustered with strains from France (MK732472.1) and from Sweden (MK458687.1) which was detected in human cerebrospinal fluid.
The Borrelia afzelii sequences in this study clustered in different branches and had significant similarities with German strains (GenBank: MK945805.1). Borrelia burgdorferi detected in Am. testudinarium clustered with strains detected in Ixodes pacificus (MN110090.1, MN110091.1, MN110092.1) in the USA. B. burgdorferi was simultaneously isolated from camel blood samples clustered with a strain isolated from humans (KM269456.1) in the USA. B. crocidurae found in camel blood clustered with other strains found in a person from France (LT984797.1) and a soft tick from Senegal (KF176328.1) (Figure 2). The presence of B. miyamotoi in ticks and camel blood samples was confirmed using glpQ sequence analysis (Figure 3). When compared to each other, most of the studied sequences were 100% identical. They shared a high level of similarity with B. miyamotoi sequences found in Ixodes persulcatus from Russia (LC538351.1), China (LC557152.1), and Japan (AB900798.1) (Asian type), as well as human blood from Russia (MK955928.1) and China (LC557152.1) (MW319188.1). The Borrelia miyamotoi sequence identified in Egyptian camels in Am. cohaerens was determined to be identical to B. miyamotoi sequences found in Ixodes ricinus from Italy (MG451835.1), and Hungary (MF678599.1), as well as in a human CSF fluid from Sweden (MK458689.1) (European type).
A list of representative sequences with GenBank accession numbers and their source (tick species or blood) is given in (Table 5). The presence of B. miyamotoi in ticks and camel blood samples was confirmed using glpQ sequence analysis (Figure 3). When compared to each other, most of the studied sequences were 100% identical. They shared a high level of similarity with B. miyamotoi sequences found in Ixodes persulcatus from Russia (LC538351.1), China (LC557152.1), and Japan (AB900798.1) (Asian type), as well as human blood from Russia (MK955928.1) and China (LC557152.1) (MW319188.1). The Borrelia miyamotoi sequence identified in Egyptian camels in Am. cohaerens was determined to be identical to B. miyamotoi sequences found in Ixodes ricinus from Italy (MG451835.1), and Hungary (MF678599.1), as well as in a human CSF fluid from Sweden (MK458689.1) (European type).

Discussion
Ticks and tick-borne diseases (TBDs) have emerged as major public health issues in many countries, including developed ones [41]. Tick infestation in camels has a high eco- A list of representative sequences with GenBank accession numbers and their source (tick species or blood) is given in (Table 5).

Discussion
Ticks and tick-borne diseases (TBDs) have emerged as major public health issues in many countries, including developed ones [41]. Tick infestation in camels has a high economic cost because ticks considerably impact their health and productivity [42]. As the prevalence of tick-borne diseases rises, distinguishing tick species is more critical than ever to improve tick and TBD control [43]. Standard morphological identification might be difficult in blood-engorged, immature, or physically injured specimens. As a result, molecular analysis can aid in discovering new information regarding ticks [44,45]. In this study, an assessment of 133 camels with an ixodid tick infestation revealed 12 species of ticks from 3 genera-infested dromedary camels. There were four species in the Hyalomma genus, six in the Amblyomma genus, and two in the Rhipicephalus genus. The genus Hyalomma was the most frequent tick genus in this study (85.1%). H. dromedarii is the most common tick species in Egyptian camels, whether locally raised or imported, according to previous studies [10,11,46,47]. H. dromedarii infection in camels is dangerous to their health, resulting in significant loss of camels and other animal products in the Middle East and North Africa [48,49], and impacts human health to some extent. Some of the tick species collected in this study (Am. lepidum, Am. variegatum, H. excavatum, R. pulchellus, and Am. gemma) are not indigenous to Egypt. This was observed in other studies [10,46,47]. These results might be attributed to camel imports to Egypt's marketplaces coming from Sudan, Ethiopia, Nigeria, and Somalia [10], suggesting that these camels could be transporting non-endemic tick species to Egypt.
GenBank BLAST using the sequences of the cox1 genes, validated tick species identification for three genera: Hyalomma, Amblyomma, and Rhipicephalus, and was identical to tick species in the GenBank database. According to the phylogenetic analysis (Figure 1), the cox1 gene is a valuable and accurate tick species identification marker [50]. Lyme disease is one of the most serious zoonotic diseases, with endemic areas in Central Asia, the United States, and Eastern Europe [51]. This disease has been neglected in the Egyptian camel population.
In the present study, the prevalence rate of Borrelia spp. (B. burgdorferi, B. miyamotoi, and B. crocidurae) was detected in camel blood samples using nested PCRs based on the IGS (16S-23S rRNA) gene (8.3%, 11/133). This result was greater than that found in a study of dromedary camels from Tunisia (1.3%, 3/232) [52] and Bactrian camel blood examined in China (3.6%, 5/138) [20]. In contrast, Borrelia spirochete DNA was not found in Iran because the bacteria does not remain in the blood for prolonged periods after infection [53]. In addition, the total prevalence of Borrelia spp. in ticks was 1.3% (21/1596). This result was lower than the prevalence of Borrelia spp. in Ixodes ticks from northern Germany (31.6%, 3150 individual ticks) [54]. While in Ethiopian soft ticks the Prevalence of Borrelia spp. was 3.5% (11/312) [55].
This investigation found Borrelia afzelii in H. dromedarii and H. marginatum. The B. afzelii sequences found in this study were very similar to isolated strains from Germany, where B. afzelii is the most common genospecies in Europe [59]. In Europe, rodents serve as reservoir hosts for B. afzelii (e.g., mice and voles) [60]. This infection causes Lyme disease, characterized by acrodermatitis chronica atrophicans and chronic skin disease [61].
While Borrelia crocidurae was found in one blood sample, it usually causes a tickborne relapsing fever (TBRF) transmitted to humans through the bites of soft ticks of the genus Ornithodoros. Its impact on public health is just being realized, and it remains an unrecognized and neglected disease [62,63]. These findings suggest that these camels and associated ticks may be infected with Lyme borrelia and other Borrelia species, posing a risk to humans.
The presence of B. burgdorferi was confirmed using 16S rRNA gene sequence analysis. Only one sample of B. burgdorferi was detected in camel blood and isolated from two H. dromedarii and one Am. testudinarium. According to the phylogenetic analysis, the Egyptian and United States strains are related. This could be related to bird migration, which aids in the spread of Lyme disease by dispersing B. burgdorferi-infected ticks across the country and introduces a new endemic foci [64,65].
TBRF is underdiagnosed in tropical areas due to diagnostic confusion with malaria [66]. B. miyamotoi is a spirochete that causes a relapsing fever; spread worldwide by hard Ixodes tick species. In 1994, B. miyamotoi was isolated from Ixodes persulcatus and in 2011, human B. miyamotoi illness was first identified in Russia [67]. It is frequently recognized as a human pathogen [28,31]. However, no human B. miyamotoi infections have been documented in Egypt. In our study, B. miyamotoi was identified in 6.8% of the camel blood samples using the glpQ gene.
B. miyamotoi has been found in six hard tick species, including H. dromedarii, Am. hebraeum, Am. lipidium, Am. variegatum, Am. cohaerens and R. annulatus. The prevalence of B. miyamotoi in ticks (1%) is comparable to that discovered in Northeast China (1.3% of 774 ticks) with rates of 2.6% in I. persulcatus, 0.78% in Dermacentor nuttalli, 1.3% in D. silvarum, and 0.4% in Haemaphysalis longicornis. [28], but the prevalence is lower than that reported from Slovak Republic rodent-attached ticks (3.4%, 31/900) [68]. B. miyamotoi DNA was detected in the blood of one camel and in the H. dromedarii that infested this camel during our investigation. Sequencing using accession numbers OL347931 and OL439927 corroborated the findings. The results indicate that Borrelia was transmitted to the tick during camel bacteremia or that a Borrelia-infected tick transmitted the infection to this camel during a blood meal [69]. In the results of our study's sequences of the glpQ gene, our B. miyamotoi was genotyped into Asian and European types and was highly similar to sequences of the gene amplified from ticks and human patients. Hence, individuals in Egypt are at risk of developing relapsing fever transmitted by ticks. Unfortunately, the role of camels is still unknown. Similarly, the competence of ticks as vectors for this pathogen needs to be confirmed. More research is needed to obtain more reliable information on the role of camels and associate ticks in pathogen transmission.

Conclusions
In conclusion, this study sheds light on the tick species that feed on camels in Egypt and demonstrates the value of cox1-based molecular approaches for identifying tick species. The camels in Egypt harbor several neglected, emerging, and re-emerging TBDs, many of which are likely new to Egypt, where Borrelia miyamotoi and Borrelia afzelii were detected for the first time in both camel blood and ticks. Future research needs to comprehend the role of camels in the enzootic cycle of Lyme borrelia. Informed Consent Statement: Informed consent was obtained from all animal owners involved in the study.
Data Availability Statement: All the data generated or analyzed in this study are included in this published article.